Inhibitors of macrophage migration inhibitory factor and methods for identifying the same

ABSTRACT

Inhibitors of MIF are provided which have utility in the treatment of a variety of disorders, including the treatment of pathological conditions associated with MIF activity. The inhibitors of MIF have the following structures: 
                         
including stereoisomers, prodrugs and pharmaceutically acceptable salts thereof, wherein n, R 1 , R 2 , R 3 , R 4 , X, and Z are as defined herein. Compositions containing an inhibitor of MIF in combination with a pharmaceutically acceptable carrier are also provided, as well as methods for use of the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 10/778,884filed Feb. 13, 2004 now U.S. Pat. No. 7,173,036, which claims thebenefit of U.S. Provisional Application No. 60/448,427, filed Feb. 14,2003.

FIELD OF THE INVENTION

This invention relates generally to inhibitors of macrophage migrationinhibitory factor (MIF), methods for identifying MIF inhibitors, and tomethods of treating MIF-related disorders by administration of suchinhibitors.

BACKGROUND OF THE INVENTION

The lymphokine, macrophage migration inhibitory factor (MIF), has beenidentified as a mediator of the function of macrophages in host defenseand its expression correlates with delayed hypersensitivity,immunoregulation, inflammation, and cellular immunity. See Metz andBucala, Adv. Immunol. 66:197–223, 1997. Macrophage migration inhibitoryfactors (MIFs), which are between 12–13 kilodaltons (kDa) in size, havebeen identified in several mammalian and avian species; see, forexample, Galat et al., Fed. Eur. Biochem. Soc. 319:233–236, 1993; Wistowet al., Proc. Natl. Acad. Sci. USA 90:1272–1275, 1993; Weiser et al.,Proc. Natl. Acad. Sci. USA 86:7522–7526, 1989; Bernhagen et al., Nature365:756–759, 1993; Blocki et al., Protein Science 2:2095–2102, 1993; andBlocki et al., Nature 360:269–270, 1992. MIF inhibitors are alsodisclosed in copending U.S. patent application Ser. No. 10/156,650 filedMay 24, 2002, the contents of which is hereby incorporated by referencein its entirety.

Although MIF was first characterized as being able to block macrophagemigration, MIF also appears to effect macrophage adherence; inducemacrophage to express interleukin-1-beta, interleukin-6, and tumornecrosis factor alpha; up-regulate HLA-DR (Human Leucocyte Antigen,d-Related, encoded by the d locus on chromosome 6 and found on lymphoidcells); increase nitric oxide synthase and nitric oxide concentrations;and activate macrophage to kill Leishmania donovani tumor cells andinhibit Mycoplasma avium growth, by a mechanism different from thateffected by interferon-gamma. In addition to its potential role as animmunoevasive molecule, MIF can act as an immunoadjuvant when given withbovine serum albumin or HIV gp120 in incomplete Freunds or liposomes,eliciting antigen induced proliferation comparable to that of completeFreunds. Also, MIF has been described as a glucocorticoid counterregulator and angiogenic factor. As one of the few proteins that isinduced and not inhibited by glucocorticoids, it serves to attenuate theimmunosuppressive effects of glucocorticoids. As such, it is viewed as apowerful element that regulates the immunosuppressive effects ofglucocorticoids. Hence, when its activities/gene expression areoverinduced by the administration of excess exogenous glucocorticoids(for example when clinical indicated to suppress inflammation, immunityand the like), there is significant toxicity because MIF itselfexacerbates the inflammatory/immune response. See Buccala et al., Ann.Rep. Med. Chem. 33:243–252, 1998.

While MIF is also thought to act on cells through a specific receptorthat in turn activates an intracellular cascade that includes erkphosphorylation and MAP kinase and upregulation of matrixmetalloproteases, c-jun (the protooncogene jun), c-fos (theprotooncogene fos) and IL-1 mRNA (see Onodera et al., J. Biol. Chem.275:444–450, 2000), it also possesses endogenous enzyme activity asexemplified by its ability to tautomerize the appropriate substrates(e.g., dopachrome). Further, it remains unclear whether this enzymaticactivity mediates the biological response to MIF and the activities ofthis protein in vitro and in vivo. While site directed mutagenesis ofMIF has generated mutants which possess full intrinsic activity, yetfail to possess enzyme activity (Hermanowski-Vosatka et al.,Biochemistry 38:12841–12849, 1999), Swope et al. have described a directlink between cytokine activity and the catalytic site for MIF (Swope etal., EMBO J. 17(13):3534–3541, 1998). Accordingly, it is unclear thatstrategies to identify inhibitors of MIF activity through inhibition ofdopachrome tautomerase alone yields inhibitors of MIF activity ofclinical value. The ability to evaluate the inhibition of MIF to itscell surface receptor is also limited since no high affinity receptor iscurrently known.

The interest in developing MIF inhibitors derives from the observationthat MIF is known for its cytokine activity concentrating macrophages atsites of infection, and cell-mediated immunity. Moreover, MIF is knownas a mediator of macrophage adherence, phagocytosis, and tumoricidalactivity. See Weiser et al., J. Immunol. 147:2006–2011, 1991. Hence, theinhibition of MIF results in the indirect inhibition of cytokines,growth factors, chemokines and lymphokines that the macrophage mayotherwise bring to a site of inflammation. Human MIF cDNA has beenisolated from a T-cell line, and encodes a protein having a molecularmass of about 12.4 kDa with 115 amino acid residues that form ahomotrimer as the active form (Weiser et al., Proc. Natl. Acad. Sci. USA86:7522–7526, 1989). While MIF was originally observed in activatedT-cells, it has now been reported in a variety of tissues including theliver, lung, eye lens, ovary, brain, heart, spleen, kidney, muscle, andothers. See Takahashi et al., Microbiol. Immunol 43(1):61–67, 1999.Another characteristic of MIF is its lack of a traditional leadersequence (i.e., a leaderless protein) to direct classical secretionthrough the Endoplasmic Reticulum/Golgi (ER/Golgi) pathway.

A MIF inhibitor (and a method to identify MIF inhibitors) that act byneutralizing the cytokine activity of MIF presents significantadvantages over other types of inhibitors. For example, the link betweentautomerase activity alone and the inflammatory response iscontroversial. Furthermore, inhibitors that act intracellularly areoften toxic by virtue of their action on related targets or theactivities of the target inside cells. Small molecule inhibitors of theligand receptor complex are difficult to identify let alone optimize anddevelop. The ideal inhibitor of a cytokine like MIF is one that altersMIF itself so that when released from the cell it is effectivelyneutralized. A small molecule with this activity is superior toantibodies because of the fundamental difference between proteins andchemicals as drugs. MIF inhibitors are disclosed in copending U.S.patent application Ser. No. 10/156,650 filed May 24, 2002.

SUMMARY OF THE INVENTION

As MIF has been identified in a variety of tissues and has beenassociated with numerous pathological events, there exists a need in theart to identify inhibitors of MIF. There is also a need forpharmaceutical compositions containing such inhibitors, as well asmethods relating to the use thereof to treat, for example, immunerelated disorders or other MIF induced pathological events, such astumor associated angiogenesis. The preferred embodiments may fulfillthese needs, and provide other advantages as well.

In preferred embodiments, inhibitors of MIF are provided that have thefollowing general structures (Ia) and (Ib):

including stereoisomers, prodrugs, and pharmaceutically acceptable saltsthereof, wherein n, R₁, R₂, R₃, R₄, X, and Z are as defined below.

The MIF inhibitors of preferred embodiments have utility over a widerange of therapeutic applications, and may be employed to treat avariety of disorders, illnesses, or pathological conditions including,but not limited to, a variety of immune related responses, tumor growth(e.g., prostate cancer, and the like), glomerulonephritis, inflammation,malarial anemia, septic shock, tumor associated angiogenesis,vitreoretinopathy, psoriasis, graft versus host disease (tissuerejection), atopic dermatitis, rheumatoid arthritis, inflammatory boweldisease, otitis media, Crohn's disease, acute respiratory distresssyndrome, delayed-type hypersensitivity, and others. See, e.g., Metz andBucala (supra); Swope and Lolis, Rev. Physiol. Biochem. Pharmacol139:1–32, 1999; Waeber et al., Diabetes M. Res. Rev. 15(1):47–54, 1999;Nishihira, Int. J. Mol. Med. 2(1):17–28, 1998; Bucala, Ann. N.Y. Acad.Sci. 840:74–82, 1998; Bernhagen et al., J. Mol. Med. 76(3–4):151–161,1998; Donnelly and Bucala, Mol. Med. Today 3(11):502–507, 1997; Bucalaet al., FASEB J. 10(14):1607–1613, 1996. Such methods includeadministering an effective amount of one or more inhibitors of MIF asprovided by the preferred embodiments, preferably in the form of apharmaceutical composition, to an animal in need thereof. Accordingly,in another embodiment, pharmaceutical compositions are providedcontaining one or more inhibitors of MIF of preferred embodiments incombination with a pharmaceutically acceptable carrier and/or diluent.

One strategy of a preferred embodiment characterizes molecules thatinteract with MIF so as to induce a conformational change in MIF and assuch a loss of immunoreactivity to a monoclonal antibody. This change,when identified by screening, identifies small molecule inhibitors ofMIF. This particular aspect may be extended to any bioactive polypeptidewhere loss of immunoreactivity may act as a surrogate for activity(e.g., cytokine activity, enzymatic activity, co-factor activity, or thelike).

In a first embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; n is 0,1, or 2, with the proviso that when n is 0, Z is —C(═O)—; R₁ is selectedfrom the group consisting of hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; R₂ and R₃ are independentlyselected from the group consisting of halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ is selected from the group consisting of:

—CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅ and R₆ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; or R₅ and R₆taken together with a nitrogen atom to which they are attached form aheterocycle or substituted heterocycle; R₇ is selected from the groupconsisting of alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; and R₈ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

In an aspect of the first embodiment, a composition is providedcomprising the compound of the first embodiment in combination with apharmaceutically acceptable carrier or diluent.

In an aspect of the first embodiment, a method is provided for treatinginflammation in a warm-blooded animal, comprising administering to theanimal an effective amount of the compound of the first embodiment.

In an aspect of the first embodiment, a method is provided for treatingseptic shock in a warm-blooded animal, comprising administering to theanimal an effective amount of the compound of the first embodiment.

In an aspect of the first embodiment, a method is provided for treatingarthritis in a warm-blooded animal, comprising administering to theanimal an effective amount of the compound of the first embodiment.

In an aspect of the first embodiment, a method is provided for treatingcancer in a warm-blooded animal, comprising administering to the animalan effective amount of the compound of the first embodiment.

In an aspect of the first embodiment, a method is provided for treatingacute respiratory distress syndrome in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of thefirst embodiment.

In an aspect of the first embodiment, a method is provided for treatingan inflammatory disease in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of thefirst embodiment. The inflammatory disease can include rheumatoidarthritis, osteoarthritis, inflammatory bowel disease, or asthma.

In an aspect of the first embodiment, a method is provided for treatingan autoimmune disorder in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of thefirst embodiment. The autoimmune disorder can include diabetes, asthma,or multiple sclerosis.

In an aspect of the first embodiment, a method is provided forsuppressing an immune response in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of thefirst embodiment.

In an aspect of the first embodiment, a method is provided fordecreasing angiogenesis in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of thefirst embodiment.

In an aspect of the first embodiment, a method is provided for treatinga disease associated with excess glucocorticoid levels in a warm-bloodedanimal, comprising administering to the animal an effective amount ofthe compound of the first embodiment. The disease can include Cushing'sdisease.

In an aspect of the first embodiment, a pharmaceutical composition isprovided for treating a disease or disorder wherein MIF is pathogenic,the pharmaceutical composition comprising a MIF inhibiting compoundaccording to the first embodiment and a drug for treating the disease ordisorder, wherein the drug has no measurable MIF inhibiting activity.

In an aspect of the first embodiment, a pharmaceutical composition isprovided for treating a disease or disorder wherein MIF is pathogenic,the pharmaceutical composition comprising a MIF inhibiting compoundaccording to the first embodiment and a drug selected from the groupconsisting of nonsteroidal anti-inflammatory drugs, anti-infectivedrugs, beta stimulants, steroids, antihistamines, anticancer drugs,asthma drugs, sepsis drugs, arthritis drugs, and immunosuppresive drugs.

In a second embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof.

In a third embodiment, a method is provided for reducing MIF activity ina patient in need thereof, comprising administering to the patient aneffective amount of a compound having the structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof.

In a fourth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein R₁ is selected from the group consisting of:

—CH₂CH₂N(R′″)₂, —CH₂CH₂NC(O)N(R′″)₂, and —CH₂COOR′″; R₁₂ is selectedfrom the group consisting of hydrogen, chlorine, fluorine, and methyl;R₄ is selected from the group consisting of:

R′″ is independently selected from the group consisting of hydrogen,fluorine, chlorine, linear C₁–C₅ alkyl, and branched C₁–C₅ alkyl; andR″″ is selected from the group consisting of hydrogen, halogen, alkyl,cyano, nitro, —COOR′″, —N(R′″)₂, —OR′″, —NHCOR′″, and —OCF₃.

In a fifth embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein R₁ is selected from the group consisting of:

R₄ is selected from the group consisting of:R₁₂ is selected from the group consisting of hydrogen, chlorine,fluorine, and methyl.

In a sixth embodiment, a method is provided for reducing MIF activity ina patient in need thereof, comprising administering to the patient aneffective amount of a compound having the structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; n is 0,1, or 2, with the proviso that when n is 0, Z is —C(═O)—; R₁ is selectedfrom the group consisting of hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; R₂ and R₃ are independentlyselected from the group consisting of halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ is selected from the group consisting of:

—CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅ and R₆ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; or R₅ and R₆taken together with a nitrogen atom to which they are attached form aheterocycle or substituted heterocycle; R₇ is selected from the groupconsisting of alkyl, substituted alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; and R₈ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

In a seventh embodiment, a compound is provided having a structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; n is 0,1, or 2, with the proviso that when n is 0, Z is —C(═O)—; R₁ is selectedfrom the group consisting of hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; R₂ and R₃ are independentlyselected from the group consisting of halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ is selected from the group consisting of:

—CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇, and R₈; R₅ and R₆ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; R₇ isselected from the group consisting of alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; and R₈ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

In an aspect of the seventh embodiment, a composition is providedcomprising the compound of the seventh embodiment in combination with apharmaceutically acceptable carrier or diluent.

In an aspect of the seventh embodiment, a method is provided fortreating inflammation in a warm-blooded animal, comprising administeringto the animal an effective amount of the compound of the seventhembodiment.

In an aspect of the seventh embodiment, a method is provided fortreating septic shock in a warm-blooded animal, comprising administeringto the animal an effective amount of the compound of the seventhembodiment.

In an aspect of the seventh embodiment, a method is provided fortreating arthritis in a warm-blooded animal, comprising administering tothe animal an effective amount of the compound of the seventhembodiment.

In an aspect of the seventh embodiment, a method is provided fortreating cancer in a warm-blooded animal, comprising administering tothe animal an effective amount of the compound of the seventhembodiment.

In an aspect of the seventh embodiment, a method is provided fortreating acute respiratory distress syndrome in a warm-blooded animal,comprising administering to the animal an effective amount of thecompound of the seventh embodiment.

In an aspect of the seventh embodiment, a method is provided fortreating an inflammatory disease in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of theseventh embodiment. The inflammatory disease can include rheumatoidarthritis, osteoarthritis, inflammatory bowel disease, or asthma.

In an aspect of the seventh embodiment, a method is provided fortreating an autoimmune disorder in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of theseventh embodiment. The autoimmune disorder can include diabetes,asthma, or multiple sclerosis.

In an aspect of the seventh embodiment, a method is provided forsuppressing an immune response in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of theseventh embodiment.

In an aspect of the seventh embodiment, a method is provided fordecreasing angiogenesis in a warm-blooded animal, comprisingadministering to the animal an effective amount of the compound of theseventh embodiment.

In an aspect of the seventh embodiment, a method is provided fortreating a disease associated with excess glucocorticoid levels in awarm-blooded animal, comprising administering to the animal an effectiveamount of the compound of the seventh embodiment. The disease caninclude Cushing's disease.

In an aspect of the seventh embodiment, a pharmaceutical composition isprovided for treating a disease or disorder wherein MIF is pathogenic,the pharmaceutical composition comprising a MIF inhibiting compoundaccording to the seventh embodiment and a drug for treating the diseaseor disorder, wherein the drug has no measurable MIF inhibiting activity.

In an aspect of the seventh embodiment, a pharmaceutical composition isprovided for treating a disease or disorder wherein MIF is pathogenic,the pharmaceutical composition comprising a MIF inhibiting compoundaccording to the seventh embodiment and a drug selected from the groupconsisting of nonsteroidal anti-inflammatory drugs, anti-infectivedrugs, beta stimulants, steroids, antihistamines, anticancer drugs,asthma drugs, sepsis drugs, arthritis drugs, and immunosuppresive drugs.

In an eighth embodiment, a method is provided for reducing MIF activityin a patient in need thereof, comprising administering to the patient aneffective amount of a compound having the structure:

or a stereoisomer, a prodrug, or a pharmaceutically acceptable saltthereof, wherein X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; n is 0,1, or 2, with the proviso that when n is 0, Z is —C(═O)—; R₁ is selectedfrom the group consisting of hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; R₂ and R₃ are independentlyselected from the group consisting of halogen, —R₅, —OR₅, —SR₅, and—NR₅R₆; R₄ is selected from the group consisting of —CH₂R₇, —C(═O)NR₅R₆,—C(═O)OR₇, —C(═O)R₇, and R₈; R₅ and R₆ are independently selected fromthe group consisting of hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, dialkyl,and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; or R₅ and R₆ taken togetherwith a nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is selected from the group consisting ofalkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; and R₈ isselected from the group consisting of hydrogen, alkyl, alkylaryl,substituted alkylaryl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acylalkyl, substituted acylalkyl, acylaryl, substitutedacylaryl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, heterocyclearyl, substitutedheterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl.

In a ninth embodiment, a process is provided for preparing a compound ofFormula IVa comprising the steps of reacting a compound of Formula I:

with a compound of Formula II:

thereby obtaining a compound of Formula III:

and reacting the compound of Formula III with a compound of formulaX—R₁, thereby obtaining a compound of Formula IVa:

wherein the compound of Formula IVa is suitable for use as a MIFinhibitor, and wherein: R₂ and R₃ are independently selected from thegroup consisting of halogen, —R₅, —OR₅, —SR₅, and —NR₅R₆; R₄ is selectedfrom the group consisting of —CH₂R₇, —C(═O)OR₇, —C(═O)R₇, R₈, and—C(═O)NR₅R₆; R₅ and R₆ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; or R₅ and R₆ taken togetherwith a nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is selected from the group consisting ofalkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; and R₈ isselected from the group consisting of hydrogen, alkyl, alkylaryl,substituted alkylaryl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acylalkyl, substituted acylalkyl, acylaryl, substitutedacylaryl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, heterocyclearyl, substitutedheterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; X is selectedfrom the group consisting of Cl, Br, and I; and R₁ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

In a tenth embodiment, a process is provided for preparing a compound ofFormula IVb comprising the steps of reacting a compound of Formula I:

with a compound of Formula II:

thereby obtaining a compound of Formula III:

and reacting the compound of Formula III with a compound comprisingX—R₁, wherein X is selected from the group consisting of Cl, Br, and I,and wherein R₁ is selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl, wherein x is2 to 4, thereby obtaining a compound of Formula IVb:

wherein the compound of Formula IVb is suitable for use as a MIFinhibitor.

In an eleventh embodiment, a process is provided for preparing acompound of Formula IVa comprising the steps of reacting a compound ofFormula I:

with piperazine, thereby obtaining a compound of Formula IIIa:

and thereafter reacting the compound of Formula IIIa with a compound ofthe formula R₄—C(O)—X, thereby obtaining a compound of Formula IVa:

wherein the compound of Formula IVa is suitable for use as a MIFinhibitor, and wherein R₂ and R₃ are independently selected from thegroup consisting of halogen, —R₅, —OR₅, —SR₅, and —NR₅R₆; R₄ is selectedfrom the group consisting of —CH₂R₇, —C(═O)NR₅R₆, R₈, —C(═O)OR₇,—C(═O)R₇; R₅ and R₆ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; or R₅ and R₆ taken togetherwith a nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is selected from the group consisting ofalkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; and R₈ isselected from the group consisting of hydrogen, alkyl, alkylaryl,substituted alkylaryl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acylalkyl, substituted acylalkyl, acylaryl, substitutedacylaryl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, heterocyclearyl, substitutedheterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; X is selectedfrom the group consisting of Cl, Br, and I; and R₁ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

In a twelfth embodiment, a process is provided for preparing a compoundof Formula IVb comprising the steps of reacting a compound of Formula I:

with piperazine, thereby obtaining a compound of Formula IIIa:

and thereafter reacting the compound of Formula IIIa with a compound ofthe formula R₄—C(O)—X, thereby obtaining a compound of Formula IVb:

wherein the compound of Formula IVb is suitable for use as a MIFinhibitor, and wherein R₂ and R₃ are independently selected from thegroup consisting of halogen, —R₅, —OR₅, —SR₅, and —NR₅R₆; R₄ is selectedfrom the group consisting of —CH₂R₇, —C(═O)NR₅R₆, R₈, —C(═O)OR₇,—C(═O)R₇; R₅ and R₆ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; or R₅ and R₆ taken togetherwith a nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is selected from the group consisting ofalkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; and R₈ isselected from the group consisting of hydrogen, alkyl, alkylaryl,substituted alkylaryl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acylalkyl, substituted acylalkyl, acylaryl, substitutedacylaryl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, heterocyclearyl, substitutedheterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; X is selectedfrom the group consisting of Cl, Br, and I; and R₁ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

In a thirteenth embodiment, a process is provided for preparing anintermediate compound of Formula I comprising the steps of reacting acompound of Formula Iaa:

with cyclohexanamine, thereby obtaining a compound of Formula IIIaa:

reacting the compound of Formula IIIaa with POCl₃, thereby obtaining acompound of Formula Ia:

and thereafter reacting the compound of Formula Ia with ammonium acetatein acetic acid, thereby obtaining an intermediate compound of Formula I:

wherein the compound of Formula I is suitable for use in preparing a MIFinhibitor, and wherein R₂ and R₃ are independently selected from thegroup consisting of halogen, —R₅, —OR₅, —SR₅, and —NR₅R₆; R₄ is selectedfrom the group consisting of —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇,and R₈; R₅ and R₆ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, andR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl; or R₅ and R₆ taken togetherwith a nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is selected from the group consisting ofalkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is2 to 4, and wherein R′ and R″ are independently selected from the groupconsisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; and R₈ isselected from the group consisting of hydrogen, alkyl, substitutedalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, alkylaryl, substituted alkylaryl, aryl, substitutedaryl, arylalkyl, substituted arylalkyl, acylalkyl, substitutedacylalkyl, acylaryl, substituted acylaryl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, and dialkyl; X is selectedfrom the group consisting of Cl, Br, and I; and R₁ is selected from thegroup consisting of hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, and R′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from the group consisting of hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, and dialkyl.

These and other embodiments and aspects thereof will be apparent uponreference to the following detailed description. To this end, variousreferences are set forth herein which describe in more detail certainprocedures, compounds and/or compositions, and are hereby incorporatedby reference in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides THP-1 Cell Assay data for Compound 200.

FIG. 2 provides in vitro tautomerase inhibitory activity data forCompound 200.

FIG. 3 provides in vitro tautomerase inhibitory activity data forCompound 203.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following description and examples illustrate a preferred embodimentof the present invention in detail. Those of skill in the art willrecognize that there are numerous variations and modifications of thisinvention that are encompassed by its scope. Accordingly, thedescription of a preferred embodiment should not be deemed to limit thescope of the present invention.

As an aid to understanding the preferred embodiments, certaindefinitions are provided herein.

The term “MIF activity,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to anactivity or effect mediated at least in part by macrophage migrationinhibitory factor. Accordingly, MIF activity includes, but is notlimited to, inhibition of macrophage migration, tautomerase activity(e.g., using phenylpyruvate or dopachrome), endotoxin induced shock,inflammation, glucocorticoid counter regulation, induction of thymidineincorporation into 3T3 fibroblasts, induction of erk phosphorylation andMAP kinase activity.

The term “export,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to ametabolically active process, which may or may not be energy-dependent,of transporting a translated cellular product to the cell membrane orthe extracellular space by a mechanism other than standard leadersequence directed secretion via a canonical leader sequence. Further,“export,” unlike secretion that is leader sequence-dependent, isresistant to brefeldin A (i.e., the exported protein is not transportedvia the ER/Golgi; brefeldin A is expected to have no direct effect ontrafficking of an exported protein) and other similar compounds. As usedherein, “export” may also be referred to as “non-classical secretion.”

The term “leaderless protein,” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer to aprotein or polypeptide that lacks a canonical leader sequence, and isexported from inside a cell to the extracellular environment. Leaderlessproteins in the extracellular environment refer to proteins located inthe extracellular space, or associated with the outer surface of thecell membrane. Within the context of preferred embodiments, leaderlessproteins include naturally occurring proteins, such as macrophagemigration inhibitory factor and fragments thereof as well as proteinsthat are engineered to lack a leader sequence and are exported, orproteins that are engineered to include a fusion of a leaderlessprotein, or fraction thereof, with another protein.

The term “inhibitor,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a molecule(e.g., natural or synthetic compound) that can alter the conformation ofMIF and/or compete with a monoclonal antibody to MIF and decrease atleast one activity of MIF or its export from a cell as compared toactivity or export in the absence of the inhibitor. In other words, an“inhibitor” alters conformation and/or activity and/or export if thereis a statistically significant change in the amount of MIF measured, MIFactivity or in MIF protein detected extracellularly and/orintracellularly in an assay performed with an inhibitor, compared to theassay performed without the inhibitor.

The term “binding agent,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to anymolecule that binds MIF, including inhibitors.

In general, MIF inhibitors inhibit the physiological function of MIF,and thus are useful in the treatment of diseases where MIF may bepathogenic.

In certain of the preferred embodiments, inhibitors of MIF are providedthat have the following structures (Ia) and (Ib):

including stereoisomers, prodrugs or pharmaceutically acceptable saltsthereof, wherein: X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; n is 0,1 or 2, with the proviso that when n is 0, Z is —C(═O)—; R₁ is hydrogen,alkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, or R′R″N(CH₂)_(x)—, wherein x is 2to 4, and wherein R′ and R″ are independently selected from hydrogen,alkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, or dialkyl; R₂ and R₃ are the same ordifferent and are independently, halogen, —R₅, —OR₅, —SR₅ or —NR₅R₆; R₄is —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇ or R₈; R₅ and R₆ are thesame or different and are independently hydrogen, alkyl, alkylaryl,substituted alkylaryl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acylalkyl, substituted acylalkyl, acylaryl, substitutedacylaryl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, heterocyclearyl, substitutedheterocyclearyl, dialkyl, or R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, or dialkyl; or R₅ and R₆ taken togetherwith the nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, orR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, ordialkyl; and R₈ is hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, orR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, ordialkyl.

In preferred embodiments, the groups R₁, R₂, R₃, and R₄ are attached tothe aromatic ring (as in the case of R₂ and R₃) or the heteroatom (as inthe case of R₁ and R₄) by a single bond. However, in certainembodiments, R₁, R₂, R₃, and R₄ can preferably be attached by a linkinggroup. Preferred linking groups have a carbon backbone, or a carbonbackbone wherein one or more of the backbone carbons are substitutedwith a heteroatom, such as nitrogen, oxygen, or sulfur. Particularlypreferred linkages contain ether groups, carboxyl groups, carbonylgroups, sulfide groups, sulfonyl groups, carboxamide groups, sulfonamidegroups, alkyl chains, aromatic rings, amine groups, and the like as partof the backbone. Preferred linking groups generally have a backbone of1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more atoms in length. Particularlypreferred backbones are of 1, 2, 3, 4, or 5 atoms in length. In apreferred embodiment, methods are provided for reducing MIF activity ina patient in need thereof by administering to the patient an effectiveamount of a compound having the following structure (Ia) and/or (Ib):

including stereoisomers, prodrugs or pharmaceutically acceptable saltsthereof, wherein: X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; n is 0,1 or 2, with the proviso that when n is 0, Z is —C(═O)—; R₁ is hydrogen,alkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, dialkyl, or R′R″N(CH₂)_(x)—, wherein x is 2to 4, and wherein R′ and R″ are independently selected from hydrogen,alkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, or dialkyl; R₂ and R₃ are the same ordifferent and are independently, halogen, —R₅, —OR₅, —SR₅ or —NR₅R₆; R₄is —CH₂R₇, —C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇ or R₈; R₅ and R₆ are thesame or different and are independently hydrogen, alkyl, alkylaryl,substituted alkylaryl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, acylalkyl, substituted acylalkyl, acylaryl, substitutedacylaryl, heterocycle, substituted heterocycle, heterocyclealkyl,substituted heterocyclealkyl, heterocyclearyl, substitutedheterocyclearyl, dialkyl, or R′R″N(CH₂)_(x)—, wherein x is 2 to 4, andwherein R′ and R″ are independently selected from hydrogen, alkyl,alkylaryl, substituted alkylaryl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, acylalkyl, substituted acylalkyl, acylaryl,substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl,substituted heterocyclearyl, or dialkyl; or R₅ and R₆ taken togetherwith the nitrogen atom to which they are attached form a heterocycle orsubstituted heterocycle; R₇ is alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, orR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, ordialkyl; and R₈ is hydrogen, alkyl, alkylaryl, substituted alkylaryl,aryl, substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, dialkyl, orR′R″N(CH₂)_(x)—, wherein x is 2 to 4, and wherein R′ and R″ areindependently selected from hydrogen, alkyl, alkylaryl, substitutedalkylaryl, aryl, substituted aryl, arylalkyl, substituted arylalkyl,acylalkyl, substituted acylalkyl, acylaryl, substituted acylaryl,heterocycle, substituted heterocycle, heterocyclealkyl, substitutedheterocyclealkyl, heterocyclearyl, substituted heterocyclearyl, ordialkyl. As used herein, the above terms have the following meanings.The term “alkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a straightchain or branched, noncyclic or cyclic, unsaturated or saturatedaliphatic hydrocarbon containing from one, two, three, four, five, six,seven, eight, nine, or ten carbon atoms, while the term “lower alkyl”has the same meaning as alkyl but contains from one, two, three, four,five, or six carbon atoms. Representative saturated straight chainalkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like; while saturated branched alkyls include isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, and the like. Representative saturatedcyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,—CH₂cyclopropyl, —CH₂cyclobutyl, —CH₂cyclopentyl, —CH₂cyclohexyl, andthe like; while unsaturated cyclic alkyls include cyclopentenyl andcyclohexenyl, and the like. Cyclic alkyls are also referred to as“homocyclic rings” and include di- and poly-homocyclic rings such asdecalin and adamantane. Unsaturated alkyls contain at least one doubleor triple bond between adjacent carbon atoms (referred to as an“alkenyl” or “alkynyl,” respectively). Representative straight chain andbranched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl,isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like; whilerepresentative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

The term “alkylaryl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an arylhaving at least one aryl hydrogen atom replaced with an alkyl moiety.

The term “aryl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an aromaticcarbocyclic moiety such as phenyl or naphthyl.

The term “arylalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylhaving at least one alkyl hydrogen atom replaced with an aryl moiety,such as benzyl, —CH₂(1 or 2-naphthyl), —(CH₂)₂phenyl, —(CH₂)₃phenyl,—CH(phenyl)₂, and the like.

The term “heteroaryl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an aromaticheterocycle ring of five, six, seven, eight, nine, or ten members andhaving at least one heteroatom selected from nitrogen, oxygen andsulfur, and containing at least one carbon atom, including bothmonocyclic and bicyclic ring systems. Representative heteroaryls include(but are not limited to) furyl, benzofuranyl, thiophenyl,benzothiophenyl, pyrrolyl, indolyl, isoindolyl, azaindolyl, pyridyl,quinolinyl, isoquinolinyl, oxazolyl, isooxazolyl, benzoxazolyl,pyrazolyl, imidazolyl, benzimidazolyl, thiazolyl, benzothiazolyl,isothiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl,cinnolinyl, phthalazinyl, and quinazolinyl.

The term “heteroarylalkyl,” as used herein is a broad term and is usedin its ordinary sense, including, without limitation, to refer to analkyl having at least one alkyl hydrogen atom replaced with a heteroarylmoiety, such as —CH₂pyridinyl, —CH₂pyrimidinyl, and the like.

The terms “heterocycle” and “heterocycle ring,” as used herein, arebroad terms and are used in their ordinary sense, including, withoutlimitation, to refer to a five, six, or seven membered monocyclic, or aseven, eight, nine, ten, eleven, twelve, thirteen, or fourteen memberedpolycyclic, heterocycle ring which is either saturated, unsaturated oraromatic, and which contains one, two, three, or four heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzene ringas well as tricyclic (and higher) heterocyclic rings. The heterocyclemay be attached via any heteroatom or carbon atom. Heterocycles includeheteroaryls as defined above. Thus, in addition to the aromaticheteroaryls listed above, heterocycles also include (but are not limitedto) morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl,valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heterocyclealkyl,” as used herein is a broad term and is usedin its ordinary sense, including, without limitation, to refer to analkyl having at least one alkyl hydrogen atom replaced with aheterocycle, such as —CH₂morpholinyl, and the like.

The term “heterocyclearyl,” as used herein is a broad term and is usedin its ordinary sense, including, without limitation, to refer to anaryl having at least one aryl hydrogen atom replaced with a heterocycle.

The term “acyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a group orradical of the form R—C(O)—L— wherein R is an organic group, includingbut not limited to alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, or substituted heterocyclearyl, each as herein defined,and L is R as defined above or a single bond. Examples of acyl groupsinclude moietes of formula:

wherein p is an integer selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,and higher integers, and wherein each Q is independently selected fromhydrogen and R, wherein R is an organic group, including but not limitedto alkyl, alkylaryl, substituted alkylaryl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, acylalkyl, substituted acylalkyl,acylaryl, substituted acylaryl, heterocycle, substituted heterocycle,heterocyclealkyl, substituted heterocyclealkyl, heterocyclearyl, orsubstituted heterocyclearyl, each as herein defined. In a preferredembodiment, p is 1 and each Q is hydrogen.

The term “arylacyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an acyl groupwherein the R group includes an aryl group as herein defined.

The term “alkylacyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an acyl groupwherein the R group includes an alkyl as herein defined.

The term “acylalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a group orradical of the form R—C(O)—Alk— wherein R is an organic group, includingbut not limited to alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, each as herein defined,and wherein Alk includes an alkyl moiety.

The term “acylaryl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a group orradical of the form R—C(O)—Ary— wherein R is an organic group, includingbut not limited to alkyl, alkylaryl, substituted alkylaryl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, acylalkyl,substituted acylalkyl, acylaryl, substituted acylaryl, heterocycle,substituted heterocycle, heterocyclealkyl, substituted heterocyclealkyl,heterocyclearyl, substituted heterocyclearyl, each as herein defined,and wherein Ary includes an aryl moiety.

The term “substituted,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to any ofthe above groups (e.g., alkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl, heterocycle or heterocyclealkyl) wherein at least onehydrogen atom is replaced with a substituent. In the case of a ketosubstituent (“—C(═O)—”) two hydrogen atoms are replaced. Whensubstituted, “substituents,” within the context of preferred embodiment,include halogen, hydroxy, cyano, nitro, sulfonamide, carboxamide,carboxyl, ether, carbonyl, amino, alkylamino, dialkylamino, alkyl,alkoxy, alkylthio, haloalkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heteroaryl, substituted heteroaryl,heteroarylalkyl, substituted heteroarylalkyl, heterocycle, substitutedheterocycle, heterocyclealkyl, substituted heterocyclealkyl,—NR_(a)R_(b), —NR_(a)C(═O)R_(b), —OR_(a), —NR_(a)C(═O)NR_(a)R_(b),—NR_(a)C(═O)OR_(b) —NR_(a)SO₂R_(b), —OR_(a), —C(═O)R_(a) —C(═O)OR_(a),—SH, —SR_(a), —C(═O)NR_(a)R_(b), —OC(═O)NR_(a)R_(b), —SOR_(a),—S(═O)₂R_(a), —OS(═O)₂R_(a), —S(═O)₂OR_(a), wherein R_(a) and R_(b) arethe same or different and independently hydrogen, alkyl, haloalkyl,substituted alkyl, aryl, substituted aryl, arylalkyl, substitutedarylalkyl, heteroaryl, substituted heteroaryl, heteroarylalkyl,substituted heteroarylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl or substituted heterocyclealkyl.

The term “halogen,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to fluoro,chloro, bromo and iodo.

The term “haloalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylhaving at least one hydrogen atom replaced with halogen, such astrifluoromethyl and the like.

The term “alkoxy,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylmoiety attached through an oxygen bridge (e.g., —O-alkyl) such asmethoxy, ethoxy, and the like.

The term “thioalkyl,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to an alkylmoiety attached through a sulfur bridge (e.g., —S-alkyl) such asmethylthio, ethylthio, and the like.

The term “alkylsulfonyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylmoiety attached through a sulfonyl bridge (e.g., —SO₂-alkyl) such asmethylsulfonyl, ethylsulfonyl, and the like.

The terms “alkylamino” and “dialkyl amino” as used herein, are broadterms and are used in their ordinary sense, including, withoutlimitation, to refer to one alkyl moiety or two alkyl moieties,respectively, attached through a nitrogen bridge (for example, —N-alkyl)such as methylamino, ethylamino, dimethylamino, diethylamino, and thelike.

The term “hydroxyalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylsubstituted with at least one hydroxyl group.

The term “mono- or di(cycloalkyl)methyl,” as used herein is a broad termand is used in its ordinary sense, including, without limitation, torefer to a methyl group substituted with one or two cycloalkyl groups,such as cyclopropylmethyl, dicyclopropylmethyl, and the like.

The term “alkylcarbonylalkyl,” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer toan alkyl substituted with a —C(═O)alkyl group.

The term “alkylcarbonyloxyalkyl,” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer toan alkyl substituted with a —C(═O)Oalkyl group or a —OC(═O)alkyl group.

The term “alkyloxyalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylsubstituted with an —O-alkyl group.

The term “alkylthioalkyl,” as used herein is a broad term and is used inits ordinary sense, including, without limitation, to refer to an alkylsubstituted with a —S-alkyl group.

The term “mono- or di(alkyl)amino,” as used herein is a broad term andis used in its ordinary sense, including, without limitation, to referto an amino substituted with one alkyl or with two alkyls, respectively.

The term “mono- or di(alkyl)aminoalkyl,” as used herein is a broad termand is used in its ordinary sense, including, without limitation, torefer to an alkyl substituted with a mono- or di(alkyl)amino.

The following numbering schemes are used in the context of preferredembodiments:

wherein R₁, R₂, R₃, R₄, X, Z, and n are as defined above.

Depending upon the Z moiety, representative compounds of preferredembodiments include the following structures (IIa) and (IIb) when Z ismethylene (—CH₂—) and structures (IIa) and (IIIb) when Z is carbonyl(—C(═O)—):

wherein R₁, R₂, R₃, R₇, and R₈, X, and n are as defined above.

In further embodiments, n is 0, 1, or 2 as represented by structures(IVa), (IVb), (Va), (Vb), (VIa), and (VIb), respectively:

wherein R₁, R₂, R₃, R₄, X, and Z are as defined above.

In still further embodiments, compounds of preferred embodiments havethe following structures (VIIa) and (VIIb) when X is oxygen andstructures (VIIIa) and (VIIIb) when X is sulfur:

wherein R₁, R₂, R₃, R₄, and Z are as defined above.

In a particularly preferred embodiment, the MIF inhibitors are of thestructure:

wherein R₁′ is selected from moieties of the following formulas:

wherein each R* is independently selected from hydrogen, halogen, alkyl,hydroxy, alkyloxy, nitro, amine, nitrile, carboxylic acid, carboxylicacid ester, alkyl amine, CF₃, —OCF₃, sulfonamide, and carboxamide. Inparticularly preferred embodiments, each R* in R₁′ is hydrogen, as inthe following structures:

In particularly preferred embodiments, the MIF inhibitors are of thefollowing structures:

The compounds of preferred embodiments may generally be employed as thefree acid or free base. Alternatively, the compounds of preferredembodiments may preferably be in the form of acid or base additionsalts. Acid addition salts of the free base amino compounds of preferredembodiments may be prepared by methods well known in the art, and may beformed from organic and inorganic acids. Suitable organic acids includemaleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic, acetic,oxalic, propionic, tartaric, salicylic, citric, gluconic, lactic,mandelic, cinnamic, aspartic, stearic, palmitic, glycolic, glutamic, andbenzenesulfonic acids. Suitable inorganic acids include hydrochloric,hydrobromic, sulfuric, phosphoric, and nitric acids. Base addition saltsof the free acid may similarly be prepared by methods well known in theart, and may be formed from suitable bases, such as cations chosen fromthe alkali and alkaline earth metals (e.g., lithium, sodium, potassium,magnesium, barium, or calcium) as well as the ammonium cation. The term“pharmaceutically acceptable salt” of structure (Ia) or (Ib) is intendedto encompass any and all acceptable salt forms.

The compounds of structure (Ia) and (Ib) may be prepared according tothe organic synthesis techniques known to those skilled in this field,as well as by the representative methods set forth in the Examples.

MIF as a Drug Target

Macrophage migration inhibitory factor (MIF) may be well suited foranalysis as a drug target as its activity has been implicated in avariety of pathophysiological conditions. For instance, MIF has beenshown to be a significant mediator in both inflammatory responses andcellular proliferation. In this regard, MIF has been shown to play rolesas a cytokine, a pituitary hormone, as glucocorticoid-inducedimmunomodulator, and just recently as a neuroimmunomodulator and inneuronal function. Takahashi et al., Mol. Med. 4:707–714, 1998; Bucala,Ann. N.Y. Acad. Sci. 840:74–82, 1998; Bacher et al., Mol. Med.4(4):217–230, 1998. Further, it has been recently demonstrated thatanti-MIF antibodies have a variety of uses, notably decreased tumorgrowth, along with an observed reduction in angiogenesis. Ogawa et al.,Cytokine 12(4):309–314, 2000; Metz and Bucala (supra). Accordingly,small molecules that can inhibit MIF have significant value in thetreatment of inflammatory responses, reduction of angiogenesis, viralinfection, bacterial infection, treatment of cancer (specificallytumorigenesis and apoptosis), treatment of graft versus host disease andassociated tissue rejection. A MIF inhibitor may be particularly usefulin a variety of immune related responses, tumor growth,glomerulonephritis, inflammation, malarial anemia, septic shock, tumorassociated angiogenesis, vitreoretinopathy, psoriasis, graft versus hostdisease (tissue rejection), atopic dermatitis, rheumatoid arthritis,inflammatory bowel disease, inflammatory lung disorders, otitis media,Crohn's disease, acute respiratory distress syndrome, delayed-typehypersensitivity. A MIF inhibitor may also be useful in the treatment ofstress and glucocorticoid function disorders, e.g., counter regulationof glucocorticoid action; or overriding of glucocorticoid mediatedsuppression of arachidonate release (Cys-60 based catalytic MIFoxidoreductase activity or JABI/CSNS-MIF-interaction based mechanism).

One example of the utility of a MIF inhibitor may be evidenced by thefact that following endotoxin exposure detectable serum concentrationsof MIF gradually increase during the acute phase (1–8 hours), peak at 8hours and persist during the post-acute phase (>8 hours) for up to 20hours. While not limited to any theory of operation, MIF may likely beproduced by activated T-cells and macrophages during the proinflammatorystage of endotoxin-induced shock, e.g., as part of the localizedresponse to infection. Once released by a pro-inflammatory stimulus,e.g., low concentrations of LPS, or by TNF-α and IFN-γ,macrophage-derived MIF may be the probable source of MIF produced duringthe acute phase of endotoxic shock. Both the pituitary, which releasesMIF in response to LPS, and macrophages are the probable source of MIFin the post-acute phase of endotoxic shock, when the infection is nolonger confined to a localized site. See, e.g. U.S. Pat. No. 6,080,407,incorporated herein by reference in its entirety and describing theseresults with anti-MIF antibodies.

As demonstrated herein, inhibitors of preferred embodiments inhibitlethality in mice following LPS challenge and likely attenuate IL-1β andTNF-α levels. Accordingly, a variety of inflammatory conditions may beamenable to treatment with a MIF inhibitor. In this regard, among otheradvantages, the inhibition of MIF activity and/or release may beemployed to treat inflammatory response and shock. Beneficial effectsmay be achieved by intervention at both early and late stages of theshock response. In this respect, while not limited to any theory ormechanism responsible for the protective effect of MIF inhibition,anti-MIF studies have demonstrated that introduction of anti-MIFantibodies is associated with an appreciable (up to 35–40%) reduction incirculating serum TNF-α levels. This reduction is consistent with theTNF-α-inducing activity of MIF on macrophages in vitro, and suggeststhat MIF may be responsible, in part, for the extremely high peak inserum TNF-α level that occurs 1–2 hours after endotoxin administrationdespite the fact that MIF cannot be detected in the circulation at thistime. Thus, MIF inhibition therapy may be beneficial at the early stagesof the inflammatory response.

MIF also plays a role during the post-acute stage of the shock response,and therefore, offers an opportunity to intervene at late stages whereother treatments, such as anti-TNF-α therapy, are ineffective.Inhibition of MIF can protect against lethal shock in animals challengedwith high concentrations of endotoxin (i.e., concentrations that inducerelease of pituitary MIF into the circulation), and in animalschallenged with TNF-α. Accordingly, the ability to inhibit MIF andprotect animals challenged with TNF indicates that neutralization of MIFduring the later, post-acute phase of septic shock may be efficacious.

As evidenced herein, TNF-α and IL-1β levels are correlated at least insome instances to MIF levels. Accordingly, an anti-MIF small moleculemay be useful in a variety of TNF-α and/or IL-1β associated diseasestates including transplant rejection, immune-mediated and inflammatoryelements of CNS disease (e.g., Alzheimer's, Parkinson's, multiplesclerosis, and the like), muscular dystrophy, diseases of hemostasis(e.g., coagulopathy, veno occlusive diseases, and the like), allergicneuritis, granuloma, diabetes, graft versus host disease, chronic renaldamage, alopecia (hair loss), acute pancreatitis, joint disease,congestive heart failure, cardiovascular disease (restenosis,atherosclerosis), joint disease, and osteoarthritis.

Further, additional evidence in the art has indicated that steroidswhile potent inhibitors of cytokine production actually increase MIFexpression. Yang et al., Mol. Med 4(6):413–424, 1998; Mitchell et al.,J. Biol. Chem. 274(25):18100–18106, 1999; Calandra and Bucala, Crit.Rev. Immunol. 17(1):77–88, 1997; Bucala, FASEB J. 10(14):1607–1613,1996. Accordingly, it may be of particular utility to utilize MIFinhibitors in combination with steroidal therapy for the treatment ofcytokine mediated pathophysiological conditions, such as inflammation,shock, and other cytokine-mediated pathological states, particularly inchronic inflammatory states such as rheumatoid arthritis. Suchcombination therapy may be beneficial even subsequent to the onset ofpathogenic or other inflammatory responses. For example, in the clinicalsetting, the administration of steroids subsequent to the onset ofseptic shock symptoms has proven of little benefit. See Bone et al., N.Engl. J. Med. 317: 653–658, 1987; Spring et al., N. Engl. J. Med. 311:1137–1141, 1984. Combination steroids/MIF inhibition therapy may beemployed to overcome this obstacle. Further, one of skill in the art mayunderstand that such therapies may be tailored to inhibit MIF releaseand/or activity locally and/or systemically.

Assays

The effectiveness of a compound as an inhibitor of MIF may be determinedby various assay methods. Suitable inhibitors of preferred embodimentsare capable of decreasing one or more activities associated with MIFand/or MIF export. A compound of structure (Ia) or (Ib) or any otherstructure may be assessed for activity as an inhibitor of MIF by one ormore generally accepted assays for this purpose, including (but notlimited to) the assays described below.

The assays may generally be divided into three categories, those being,assays which monitor export; those which monitor effector or smallmolecule binding, and those that monitor MIF activity. However, itshould be noted that combinations of these assays are within the scopeof the present application. Surprisingly, it appears that epitopemapping of MIF acts as surrogate for biological activity. For example,in one assay, the presence of a candidate inhibitor blocks the detectionof export of MIF from cells (e.g., THP-1 cells—a human acute monocyticleukemia cell line) measured using a monoclonal antibody such as thatcommercially available from R&D systems (Minneapolis, Minn.) whereas apolyclonal antibody demonstrates that MIF is present. Further, cellularbased or in vitro assays may be employed to demonstrate that thesepotential inhibitors inhibit MIF activity. In an alternative, these twoassays (i.e., binding and activity assays) may be combined into asingular assay which detects binding of a test compound (e.g., theability to displace monoclonal antibodies or inhibit their binding)while also affecting MIF activity. Such assays include combining anELISA type assay (or similar binding assay) with a MIF tautomerism assayor similar functional assay. As one of ordinary skill in the art mayreadily recognize, the exact assay employed is irrelevant, provided itis able to detect the ability of the compound of interest to bind MIF.In addition, the assay preferably detects the ability of the compound toinhibit a MIF activity because it selects for compounds that interactwith biologically active MIF and not inactive MIF.

It should also be understood that compounds demonstrating the ability toinhibit monoclonal antibody binding to biologically active and notinactive MIF (e.g., small molecule inhibited), necessarily indicate thepresence of a compound (e.g., a small molecule) that is interacting withMIF either in a fashion which changes the conformation of MIF or blocksan epitope necessary for antibody binding. In other embodiments, MIFinhibitory activity may also be recognized as a consequence ofinterfering with the formation of a polypeptide complex that includesMIF; disturbing such a complex may result in a conformational changeinhibiting detection. Accordingly, the use of assays that monitorconformational changes in MIF, are advantageous when employed either inaddition to assays measuring competition between compounds, such assmall molecules with mAb or as a replacement of such an assay. A varietyof such assays are known in the art and include, calorimetry,circular-dichroism, fluorescence energy transfer, light-scattering,nuclear magnetic resonance (NMR), surface plasmon resonance,scintillation proximity assays (see U.S. Pat. No. 5,246,869), and thelike. See also WO02/07720-A1 and WO97/29635-A1. Accordingly, one ofskill in the art may recognize that any assay that indicates binding andpreferably conformational change or placement near the active site ofMIF may be utilized. Descriptions of several of the more complicatedproximity assays and conformational assays are set forth below, thisdiscussion is merely exemplary and in no way should be construed aslimiting to the type of techniques that may be utilized in preferredembodiments.

In one example, circular dichroism may be utilized to determinecandidate inhibitor binding. Circular dichroism (CD) is based in part onthe fact that most biological protein macromolecules are made up ofasymmetric monomer units, L-amino acids, so that they all possess theattribute of optical activity. Additionally, rigid structures like DNAor an alpha helical polypeptide have optical properties that can bemeasured using the appropriate spectroscopic system. In fact, largechanges in an easily measured spectroscopic parameter can provideselective means to identify conformational states and changes inconformational states under various circumstances, and sometimes toobserve the perturbation of single groups in or attached to themacromolecule. Further, CD analysis has been frequently employed toprobe the interactions of various macromolecules with small moleculesand ligands. See Durand et al., Eur. Biophys. J. 27(2):147–151, 1998;Kleifeld et al., Biochem 39(26):7702–7711, 2000; Bianchi et al., Biochem38(42):13844–13852, 1999; Sarver et al., Biochim Biophys Acta1434(2):304–316, 1999.

The Pasteur principle states that an optically active molecule must beasymmetric; that is, the molecule and its mirror image cannot besuperimposed. Plane polarized light is a combination of left circularlypolarized light and right circularly polarized light traveling in phase.The interaction of this light with an asymmetric molecule results in apreferential interaction of one circularly polarized component which, inan absorption region, is seen as a differential absorption (i.e., adichroism). See Urry, D. W., Spectroscopic Approaches to BiomolecularConformation, American Medical Association Press, Chicago, Ill., pp.33–120 (1969); Berova and Woody, Circular Dichroism: Principles andApplications, John Wiley & Sons, N.Y., (2000).

Circular dichroism, then, is an absorptive phenomenon that results whena chromophore interacts with plane polarized light at a specificwavelength. The absorption band can be either negative or positivedepending on the differential absorption of the right and leftcircularly polarized components for that chromophore. Unlike opticalrotatory dispersion (ORD) that measures the contributions of backgroundand the chromophore of interest many millimicrons from the region ofactual light interaction, CD offers the advantage of measuring opticalevents at the wavelength at which the event takes place. Circulardichroism, then, is specific to the electronic transition of thechromophore. See Berova and Woody, Circular Dichroism: Principles andApplications, John Wiley & Sons, N.Y., (2000).

Application of circular dichroism to solutions of macromolecules hasresulted in the ability to identify conformation states (Berova andWoody, Circular Dichroism: Principles and Applications, John Wiley &Sons, N.Y., (2000)). The technique can distinguish random coil, alphahelix, and beta chain conformation states of macromolecules. Inproteins, alpha helical fibrous proteins show absorption curves closelyresembling those of alpha helical polypeptides, but in globular proteinsof known structure, like lysozyme and ribonuclease, the helicalstructures are in rather poor agreement with X-ray crystallography work.A further source of difficulty in globular proteins is the prevalence ofaromatic chromophores on the molecules around 280 nm. An interestingexample of helical changes has been demonstrated using myoglobin andapomyoglobin. After removing the prosthetic group heme, the apoproteinremaining has a residual circular dichroic ellipticity reduced by 25%.This loss of helix is attributable to an uncoiling of 10–15 residues inthe molecule. Other non-peptide, optically active chromophores includetyrosine, tryptophan, phenylalanine, and cysteine when located in theprimary amino acid sequence of a macromolecule. Examples of non-peptideellipticities include the disulfide transition in ribonuclease and thecysteine transitions of insulin.

Accordingly, circular dichroism may be employed to screen candidateinhibitors for the ability to affect the conformation of MIF.

In certain embodiments provided herein, MIF-binding agent or inhibitorcomplex formation may be determined by detecting the presence of acomplex including MIF and a detectably labeled binding agent. Asdescribed in greater detail below, fluorescence energy signal detection,for example by fluorescence polarization, provides determination ofsignal levels that represent formation of a MIF-binding agent molecularcomplex. Accordingly, and as provided herein, fluorescence energysignal-based comparison of MIF-binding agent complex formation in theabsence and in the presence of a candidate inhibitor provides a methodfor identifying whether the agent alters the interaction between MIF andthe binding agent. For example, the binding agent may be a MIFsubstrate, an anti-MIF antibody, or a known inhibitor, while thecandidate inhibitor may be the compound to be tested or vice versa.

As noted above, certain preferred embodiments also pertain in part tofluorescence energy signal-based determination of MIF-binding agentcomplex formation. Fluorescence energy signal detection may be, forexample, by fluorescence polarization or by fluorescence resonanceenergy transfer, or by other fluorescence methods known in the art. Asan example of certain other embodiments, the MIF polypeptide may belabeled as well as the candidate inhibitor and may comprise an energytransfer molecule donor-acceptor pair, and the level of fluorescenceresonance energy transfer from energy donor to energy acceptor isdetermined.

In certain embodiments the candidate inhibitor and/or binding agent isdetectably labeled, and in particularly preferred embodiments thecandidate inhibitor and/or binding agent is capable of generating afluorescence energy signal. The candidate inhibitor and/or binding agentcan be detectably labeled by covalently or non-covalently attaching asuitable reporter molecule or moiety, for example any of variousfluorescent materials (e.g., a fluorophore) selected according to theparticular fluorescence energy technique to be employed, as known in theart and based upon the methods described herein. Fluorescent reportermoieties and methods for as provided herein can be found, for example inHaugland (1996 Handbook of Fluorescent Probes and ResearchChemicals—Sixth Ed., Molecular Probes, Eugene, Oreg.; 1999 Handbook ofFluorescent Probes and Research Chemicals—Seventh Ed., Molecular Probes,Eugene, Oreg., http://www.probes.com/lit/) and in references citedtherein. Particularly preferred for use as such a fluorophore inpreferred embodiments are fluorescein, rhodamine, Texas Red,AlexaFluor-594, AlexaFluor-488, Oregon Green, BODIPY-FL, and Cy-5.However, any suitable fluorophore may be employed, and in certainembodiments fluorophores other than those listed may be preferred.

As provided herein, a fluorescence energy signal includes anyfluorescence emission, excitation, energy transfer, quenching, ordequenching event or the like. Typically a fluorescence energy signalmay be mediated by a fluorescent detectably labeled candidate inhibitorand/or binding agent in response to light of an appropriate wavelength.Briefly, and without wishing to be bound by theory, generation of afluorescence energy signal generally involves excitation of afluorophore by an appropriate energy source (e.g., light of a suitablewavelength for the selected fluorescent reporter moiety, or fluorophore)that transiently raises the energy state of the fluorophore from aground state to an excited state. The excited fluorophore in turn emitsenergy in the form of detectable light typically having a different(e.g., usually longer) wavelength from that preferred for excitation,and in so doing returns to its energetic ground state. The methods ofpreferred embodiments contemplate the use of any fluorescence energysignal, depending on the particular fluorophore, substrate labelingmethod and detection instrumentation, which may be selected readily andwithout undue experimentation according to criteria with which thosehaving ordinary skill in the art are familiar.

In certain embodiments, the fluorescence energy signal is a fluorescencepolarization (FP) signal. In certain other embodiments, the fluorescenceenergy signal may be a fluorescence resonance energy transfer (FRET)signal. In certain other preferred embodiments the fluorescence energysignal can be a fluorescence quenching (FQ) signal or a fluorescenceresonance spectroscopy (FRS) signal. (For details regarding FP, FRET, FQand FRS, see, for example, WO97/39326; WO99/29894; Haugland, Handbook ofFluorescent Probes and Research Chemicals—6th Ed., 1996, MolecularProbes, Inc., Eugene, Oreg., p. 456; and references cited therein.)

FP, a measurement of the average angular displacement (due to molecularrotational diffusion) of a fluorophore that occurs between itsabsorption of a photon from an energy source and its subsequent emissionof a photon, depends on the extent and rate of rotational diffusionduring the excited state of the fluorophore, on molecular size andshape, on solution viscosity and on solution temperature (Perrin, 1926J. Phys. Rad. 1:390). When viscosity and temperature are held constant,FP is directly related to the apparent molecular volume or size of thefluorophore. The polarization value is a ratio of fluorescenceintensities measured in distinct planes (e.g., vertical and horizontal)and is therefore a dimensionless quantity that is unaffected by theintensity of the fluorophore. Low molecular weight fluorophores, such asthe detectably labeled candidate inhibitor and/or binding agent providedherein, are capable of rapid molecular rotation in solution (i.e., lowanisotropy) and thus give rise to low fluorescence polarizationreadings. When complexed to a higher molecular weight molecule such asMIF as provided herein, however, the fluorophore moiety of the substrateassociates with a complex that exhibits relatively slow molecularrotation in solution (i.e., high anisotropy), resulting in higherfluorescence polarization readings.

This difference in the polarization value of free detectably labeledcandidate inhibitor and/or binding agent compared to the polarizationvalue of MIF:candidate inhibitor and/or binding agent complex may beemployed to determine the ratio of complexed (e.g., bound) to free. Thisdifference may also be employed to detect the influence of a candidateagent (i.e., candidate inhibitor) on the formation of such complexesand/or on the stability of a pre-formed complex, for example bycomparing FP detected in the absence of an agent to FP detected in thepresence of the agent. FP measurements can be performed withoutseparation of reaction components.

As noted above, one aspect of a preferred embodiment utilizes thebinding or displacement of a monoclonal antibody, known inhibitor, orother binding agent and/or complex formation of the candidate inhibitorwith MIF to provide a method of identifying an inhibitor that alters theactivity of MIF. Surprisingly, the inhibitors of preferred embodimentswere identified in such a nonconventional manner. In this regard, aclass of compounds demonstrated the ability to inhibit/decreasemonoclonal antibody binding to a biologically active MIF that isnaturally produced from cells while not affecting the antibody's abilityto recognize inactive (recombinant) MIF (as is available from commercialsources) and also demonstrated pronounced modulation of MIF activity invivo. Accordingly, antibody binding may be preferred as a surrogate forenzyme activity, thus eliminating the need to run expensive and complexenzymatic assays on each candidate compound. As those of ordinary skillin the art readily appreciate, the ability to avoid enzymatic assaysleads to an assay that may be extremely well suited for high throughputuse.

Further, as those of ordinary skill in the art can readily appreciate,such an assay may be employed outside of the MIF context and whereverenzyme or biological activity can be replaced by a binding assay. Forexample, any enzyme or other polypeptide whose biologically active formis recognized by a monoclonal antibody that does not recognize theinactive form (e.g., small molecule inhibited form) may be preferred.Within the context of an enzyme, the monoclonal antibody may bind theactive site, but be displaced by a small molecule. Thus, any smallmolecule that displaces the antibody may be a strong lead as a potentialenzyme inhibitor. As those of skill in the art appreciate, the antibodymay recognize an epitope that changes conformation depending on theactive state of the enzyme, and that binding of a small molecule suchthat it precludes antibody binding to this epitope may also act as asurrogate for enzymatic activity even though the epitope may not be atthe active site. Accordingly, the type of assay utilized herein may beexpanded to be employed with essentially any polypeptide whereinantibody displacement is predictive of activity loss. Thus, in itssimplest form any polypeptide, e.g., enzyme and its associatedneutralizing antibody may be employed to screen for small molecules thatdisplace this antibody, thereby identifying likely inhibitors.

A MIF-binding agent/candidate inhibitor complex may be identified by anyof a variety of techniques known in the art for demonstrating anintermolecular interaction between MIF and another molecule as describedabove, for example, co-purification, co-precipitation,co-immunoprecipitation, radiometric or fluorimetric assays, westernimmunoblot analyses, affinity capture including affinity techniques suchas solid-phase ligand-counterligand sorbent techniques, affinitychromatography and surface affinity plasmon resonance, NMR, and the like(see, e.g., U.S. Pat. No. 5,352,660). Determination of the presence ofsuch a complex may employ antibodies, including monoclonal, polyclonal,chimeric and single-chain antibodies, and the like, that specificallybind to MIF or the binding agent.

Labeled MIF and/or labeled binding agents/candidate inhibitors can alsobe employed to detect the presence of a complex. The molecule ofinterest can be labeled by covalently or non-covalently attaching asuitable reporter molecule or moiety, for example any of variousenzymes, fluorescent materials, luminescent materials, and radioactivematerials. Examples of suitable enzymes include, but are not limited to,horseradish peroxidase, alkaline phosphatase, β-galactosidase, andacetylcholinesterase. Examples of suitable fluorescent materialsinclude, but are not limited to, umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride, phycoerythrin, Texas Red, AlexaFluor-594, AlexaFluor-488,Oregon Green, BODIPY-FL and Cy-5. Appropriate luminescent materialsinclude, but are not limited to, luminol and suitable radioactivematerials include radioactive phosphorus [³²P], iodine [¹²⁵I or ¹³¹I] ortritium [³H].

MIF and the binding agent and/or the candidate inhibitor are combinedunder conditions and for a time sufficient to permit formation of anintermolecular complex between the components. Suitable conditions forformation of such complexes are known in the art and can be readilydetermined based on teachings provided herein, including solutionconditions and methods for detecting the presence of a complex and/orfor detecting free substrate in solution.

Association of a detectably labeled binding agent(s) and/or candidateinhibitor(s) in a complex with MIF, and/or binding agent or candidateinhibitor that is not part of such a complex, may be identifiedaccording to a preferred embodiment by detection of a fluorescenceenergy signal generated by the substrate. Typically, an energy sourcefor detecting a fluorescence energy signal is selected according tocriteria with which those having ordinary skill in the art are familiar,depending on the fluorescent reporter moiety with which the substrate islabeled. The test solution, containing (a) MIF and (b) the detectablylabeled binding agent and/or candidate inhibitor, is exposed to theenergy source to generate a fluorescence energy signal, which isdetected by any of a variety of well known instruments and identifiedaccording to the particular fluorescence energy signal. In preferredembodiments, the fluorescence energy signal is a fluorescencepolarization signal that can be detected using a spectrofluorimeterequipped with polarizing filters. In particularly preferred embodimentsthe fluorescence polarization assay is performed simultaneously in eachof a plurality of reaction chambers that can be read using an LJLCRITERION™ Analyst (LJL Biosystems, Sunnyvale, Calif.) plate reader, forexample, to provide a high throughput screen (HTS) having variedreaction components or conditions among the various reaction chambers.Examples of other suitable instruments for obtaining fluorescencepolarization readings include the POLARSTAR™ (BMG Lab Technologies,Offenburg, Germany), BEACON™ (Panvera, Inc., Madison, Wis.) and thePOLARION™ (Tecan, Inc., Research Triangle Park, N.C.) devices.

Determination of the presence of a complex that has formed between MIFand a binding agent and/or a candidate inhibitor may be performed by avariety of methods, as noted above, including fluorescence energy signalmethodology as provided herein and as known in the art. Suchmethodologies may include, by way of illustration and not limitation FP,FRET, FQ, other fluorimetric assays, co-purification, co-precipitation,co-immunoprecipitation, radiometric, western immunoblot analyses,affinity capture including affinity techniques such as solid-phaseligand-counterligand sorbent techniques, affinity chromatography andsurface affinity plasmon resonance, circular dichroism, and the like.For these and other useful affinity techniques, see, for example,Scopes, R. K., Protein Purification: Principles and Practice, 1987,Springer-Verlag, NY; Weir, D. M., Handbook of Experimental Immunology,1986, Blackwell Scientific, Boston; and Hermanson, G. T. et al.,Immobilized Affinity Ligand Techniques, 1992, Academic Press, Inc.,California; which are hereby incorporated by reference in theirentireties, for details regarding techniques for isolating andcharacterizing complexes, including affinity techniques. In variousembodiments, MIF may interact with a binding agent and/or candidateinhibitor via specific binding if MIF binds the binding agent and/orcandidate inhibitor with a K_(a) of greater than or equal to about 10⁴M⁻¹, preferably of greater than or equal to about 10⁵ M⁻¹, morepreferably of greater than or equal to about 10⁶ M⁻¹ and still morepreferably of greater than or equal to about 10⁷ M⁻¹ to 10¹¹ M⁻¹.Affinities of binding partners can be readily calculated from datagenerated according to the fluorescence energy signal methodologiesdescribed above and using conventional data handling techniques, forexample, those described by Scatchard et al., Ann. N.Y. Acad. Sci.51:660 (1949).

For example, in various embodiments where the fluorescence energy signalis a fluorescence polarization signal, fluorescence anisotropy (inpolarized light) of the free detectably labeled candidate inhibitorand/or binding agent can be determined in the absence of MIF, andfluorescence anisotropy (in polarized light) of the fully boundsubstrate can be determined in the presence of a titrated amount of MIF.Fluorescence anisotropy in polarized light varies as a function of theamount of rotational motion that the labeled candidate inhibitor and/orbinding agent undergoes during the lifetime of the excited state of thefluorophore, such that the anisotropies of free and fully boundcandidate inhibitor and/or binding agent can be usefully employed todetermine the fraction of candidate inhibitor and/or binding agent boundto MIF in a given set of experimental conditions, for instance, thosewherein a candidate agent is present (see, e.g., Lundblad et al., 1996Molec. Endocrinol. 10:607; Dandliker et al., 1971 Immunochem. 7:799;Collett, E., Polarized Light: Fundamentals and Applications, 1993 MarcelDekker, New York).

Certain of the preferred embodiments pertain in part to the use ofintermolecular energy transfer to monitor MIF-binding agent complexformation and stability and/or MIF-candidate inhibitor complexformation.

Energy transfer (ET) is generated from a resonance interaction betweentwo molecules: an energy-contributing “donor” molecule and anenergy-receiving “acceptor” molecule. Energy transfer can occur when (1)the emission spectrum of the donor overlaps the absorption spectrum ofthe acceptor and (2) the donor and the acceptor are within a certaindistance (for example, less than about 10 nm) of one another. Theefficiency of energy transfer is dictated largely by the proximity ofthe donor and acceptor, and decreases as a power of 6 with distance.Measurements of ET thus strongly reflect the proximity of the acceptorand donor compounds, and changes in ET sensitively reflect changes inthe proximity of the compounds such as, for example, association ordissociation of the donor and acceptor.

It is therefore an aspect of a preferred embodiment to provide a methodfor assaying a candidate MIF inhibitor, in pertinent part, by contactingMIF or an MIF-binding agent complex including one or more ET donor andan ET acceptor molecules, exciting the ET donor to produce an excited ETdonor molecule and detecting a signal generated by energy transfer fromthe ET donor to the ET acceptor. As also provided herein, the method canemploy any suitable ET donor molecule and ET acceptor molecule that canfunction as a donor-acceptor pair.

In certain preferred embodiments, a detectable signal that is generatedby energy transfer between ET donor and acceptor molecules results fromfluorescence resonance energy transfer (FRET), as discussed above. FREToccurs within a molecule, or between two different types of molecules,when energy from an excited donor fluorophore is transferred directly toan acceptor fluorophore (for a review, see Wu et al., AnalyticalBiochem. 218:1–13, 1994).

In other aspects of preferred embodiments, the ability of a candidateinhibitor to effect MIF export is tested.

The first step of such an assay is performed to detect MIFextracellularly. For this assay, test cells expressing MIF are employed(e.g., THP-1 cells). Either the test cells may naturally produce theprotein or produce it from a transfected expression vector. Test cellspreferably normally express the protein, such that transfection merelyincreases expressed levels. Thus, for expression of MIF and IL-1, THP1cells are preferred. When one is assaying virally-derived proteins, suchas HIV tat (a protein released from Human Immunodeficiency. Virusinfected cells), if the test cells do not “naturally” produce theprotein, they may readily be transfected using an appropriate vector, sothat the test cells express the desired protein, as those of skill inthe art readily appreciate.

Following expression, MIF is detected by any one of a variety ofwell-known methods and procedures. Such methods include staining withantibodies in conjunction with flow cytometry, confocal microscopy,image analysis, immunoprecipitation of cell cytosol or medium, Westernblot of cell medium, ELISA, 1- or 2-D gel analysis, HPLC, or bioassay. Aconvenient assay for initial screening is ELISA. MIF export may beconfirmed by one of the other assays, preferably by immunoprecipitationof cell medium following metabolic labeling. Briefly, cells expressingMIF protein are pulse labeled for 15 minutes with ³⁵S-methionine and/or³⁵S-cysteine in methionine and/or cysteine free medium and chased inmedium supplemented with excess methionine and/or cysteine. Mediafractions are collected and clarified by centrifugation, such as in amicrofuge. Lysis buffer containing 1% NP-40, 0.5% deoxycholate (DOC), 20mM Tris, pH 7.5, 5 mM ethylene diamine tetraacetic acid (EDTA), 2 mMEGTA, 10 nM phenyl methyl sulfonyl fluoride (PMSF), 10 ng/ml aprotinin,10 ng/ml leupeptin, and 10 ng/ml pepstatin is added to the clarifiedmedium. An antibody to MIF is added and following incubation in thecold, a precipitating second antibody or immunoglobulin binding protein,such as protein A-Sepharose® or GammaBind™-Sepharose®, is added forfurther incubation. A-Sepharose® is Protein A, an immunoglobulin G (IgG)binding reagent used for measurement and purification of free and cellbound antigens and antibodies, that is available from Pharmacia, Inc.Protein A binds the Fc portion of antibodies (IgG class) withoutdisturbing their binding of antigen. GammaBind™-Sepharose® fromPharmacia, Inc. is Protein G, a binding reagent that binds to theconstant region of many types of immunoglobulin G, and can be used todetect, quantify and purify IgG antibodies and antigen/antibodycomplexes. In parallel, as a control, a cytosolic protein is monitoredand an antibody to the cytosolic protein is preferred inimmunoprecipitations. Immune complexes are pelleted and washed withice-cold lysis buffer. Complexes are further washed with ice-cold IPbuffer (0.15 M NaCl, 10 mM Na-phosphate, pH 7.2, 1% DOC, 1% NP-40, 0.1%SDS). Immune complexes are eluted directly into SDS-gel sample bufferand electrophoresed in SDS-PAGE. The gel is processed for fluorography,dried and exposed to X-ray film. Alternatively cells can be engineeredto produced a MIF that is tagged with a reporter so that the presence ofan active MIF can be through the surrogate activity of the reporter.

While not wishing to be bound to theory, it is believed that the presentinhibitors function by interacting directly with the naturally producedMIF complex in such a fashion as to alter the protein's conformationenough to block its biological activity. This interaction can be mappedby X-ray crystallography of MIF-compound co-crystals to determine theexact site of interaction. One site localizes to the pocket that isresponsible for the tautomerase activity of MIF.

Screening assays for inhibitors of MIF export varies according to thetype of inhibitor and the nature of the activity that is being affected.Assays may be performed in vitro or in vivo. In general, in vitro assaysare designed to evaluate MIF activity, or multimerization, and in vivoassays are designed to evaluate MIF activity, extracellular, andintracellular localization in a model cell or animal system. In any ofthe assays, a statistically significant increase or decrease compared toa proper control is indicative of enhancement or inhibition.

One in vitro assay can be performed by examining the effect of acandidate compound on the ability of MIF to inhibit macrophagemigration. Briefly, human peripheral blood monocytes are preferred asindicator cells in an agarose-droplet assay system essentially asdescribed by Weiser et al., Cell. Immunol. 90:167–178, 1985 andHarrington et al., J. Immunol. 110:752–759, 1973. Other assay systems ofanalyzing macrophage migration may also be employed. Such an assay isdescribed by Hermanowski-Vosatka et al., Biochem. 38:12841–12849, 1999.

An alternative in vitro assay is designed to measure the ability of MIFto catalyze tautomerization of the D-isomer of dopachrome (see Rosengrenet al., Mol. Med 2:143–149, 1996; Winder et al., J. Cell Sci.106:153–166, 1993; Aroca et al., Biochem. J. 277:393–397). Briefly, inthis method, D-dopachrome is provided to MIF in the presence and absenceof a candidate inhibitor and the ability to catalyze the tautomerizationto 5,6-dihydroxyindole-2-carboxylic acid (DHICA) is monitored. However,use of methyl esters of D-dopachrome may be preferred in that a fasterreaction rate is observed. Detection of the tautomerization can beperformed by any one of a variety of standard methods.

In a similar assay, the ability of MIF to catalyze the tautomerizationof phenylpyruvate may be tested (see Johnson et al., Biochem.38(48):16024–16033, 1999). Briefly, in this method, typicallyketonization of phenylpyruvate or (p-hydroxyphenyl)pyruvate is followedby spectroscopy. Further, product formation may be verified by treatmentof these compounds with MIF and subsequent conversion to malate for ¹HNMR analysis.

In vivo assays can be performed in cells transfected either transientlyor stably with an expression vector containing a MIF nucleic acidmolecule, such as those described herein. These cells are preferred tomeasure MIF activity (e.g., modulation of apoptosis, proliferation, andthe like) or extracellular and intracellular localization in thepresence or absence of a candidate compound. When assaying forapoptosis, a variety of cell analyses may be employed including, forexample, dye staining and microscopy to examine nucleic acidfragmentation and porosity of the cells.

Other assays may be performed in model cell or animal systems, byproviding to the system a recombinant or naturally occurring form of MIFor inducing endogenous MIF expression in the presence or absence of testcompound, thereby determining a statistically significant increase ordecrease in the pathology of that system. For example, LPS can beemployed to induce a toxic shock response.

The assays briefly described herein may be employed to identify aninhibitor that is specific for MIF.

In any of the assays described herein, a test cell may express the MIFnaturally (e.g., THP-1 cells) or following introduction of a recombinantDNA molecule encoding the protein. Transfection and transformationprotocols are well known in the art and include Ca₂PO₄-mediatedtransfection, electroporation, infection with a viral vector,DEAE-dextran mediated transfection, and the like. As an alternative tothe proteins described above, chimeric MIF proteins (i.e., fusion of MIFprotein with another protein or protein fragment), or protein sequencesengineered to lack a leader sequence may be employed. In a similarfashion, a fusion may be constructed to direct secretion, export, orcytosolic retention. Any and all of these sequences may be employed in afusion construct with MIF to assist in assaying inhibitors. The hostcell can also express MIF as a result of being diseased, infected with avirus, and the like. Secreted proteins that are exported by virtue of aleader sequence are well known and include, human chorionic gonadatropin(hCGα), growth hormone, hepatocyte growth factor, transferrin, nervegrowth factor, vascular endothelial growth factor, ovalbumin, andinsulin-like growth factor. Similarly, cytosolic proteins are well knownand include, neomycin phosphotransferase, β-galactosidase, actin andother cytoskeletal proteins, and enzymes, such as protein kinase A or C.The most useful cytosolic or secreted proteins are those that arereadily measured in a convenient assay, such as ELISA. The threeproteins (leaderless, secreted, and cytosolic) may be co-expressednaturally, by co-transfection in the test cells, or transfectedseparately into separate host cells. Furthermore, for the assaysdescribed herein, cells may be stably transformed or express the proteintransiently.

Immunoprecipitation is one such assay that may be employed to determineinhibition. Briefly, cells expressing MIF naturally or from anintroduced vector construct are labeled with ³⁵S-methionine and/or³⁵S-cysteine for a brief period of time, typically 15 minutes or longer,in methionine- and/or cysteine-free cell culture medium. Following pulselabeling, cells are washed with medium supplemented with excessunlabeled methionine and cysteine plus heparin if the leaderless proteinis heparin binding. Cells are then cultured in the same chase medium forvarious periods of time. Candidate inhibitors or enhancers are added tocultures at various concentration. Culture supernatant is collected andclarified. Supernatants are incubated with anti-MIF immune serum or amonoclonal antibody, or with anti-tag antibody if a peptide tag ispresent, followed by a developing reagent such as Staphylococcus aureusCowan strain I, protein A-Sepharose®, or Gamma-bind™ G-Sepharose®.Immune complexes are pelleted by centrifugation, washed in a buffercontaining 1% NP-40 and 0.5% deoxycholate, EGTA, PMSF, aprotinin,leupeptin, and pepstatin. Precipitates are then washed in a buffercontaining sodium phosphate pH 7.2, deoxycholate, NP-40, and SDS. Immunecomplexes are eluted into an SDS gel sample buffer and separated bySDS-PAGE. The gel is processed for fluorography, dried, and exposed tox-ray film.

Alternatively, ELISA may be preferred to detect and quantify the amountof MIF in cell supernatants. ELISA is preferred for detection in highthroughput screening. Briefly, 96-well plates are coated with ananti-MIF antibody or anti-tag antibody, washed, and blocked with 2% BSA.Cell supernatant is then added to the wells. Following incubation andwashing, a second antibody (e.g., to MIF) is added. The second antibodymay be coupled to a label or detecting reagent, such as an enzyme or tobiotin. Following further incubation, a developing reagent is added andthe amount of MIF determined using an ELISA plate reader. The developingreagent is a substrate for the enzyme coupled to the second antibody(typically an anti-isotype antibody) or for the enzyme coupled tostreptavidin. Suitable enzymes are well known in the art and includehorseradish peroxidase, which acts upon a substrate (e.g., ABTS)resulting in a colorimetric reaction. It is recognized that rather thanusing a second antibody coupled to an enzyme, the anti-MIF antibody maybe directly coupled to the horseradish peroxidase, or other equivalentdetection reagent. If desired, cell supernatants may be concentrated toraise the detection level. Further, detection methods, such as ELISA andthe like may be employed to monitor intracellular as well asextracellular levels of MIF. When intracellular levels are desired, acell lysate is preferred. When extracellular levels are desired, mediacan be screened.

ELISA may also be readily adapted for screening multiple candidateinhibitors or enhancers with high throughput. Briefly, such an assay isconveniently cell based and performed in 96-well plates. Test cells thatnaturally or stably express MIF are plated at a level sufficient forexpressed product detection, such as, about 20,000 cells/well. However,if the cells do not naturally express the protein, transient expressionis achieved, such as by electroporation or Ca₂PO₄-mediated transfection.For electroporation, 100 μl of a mixture of cells (e.g., 150,000cells/ml) and vector DNA (5 μg/ml) is dispensed into individual wells ofa 96-well plate. The cells are electroporated using an apparatus with a96-well electrode (e.g., ECM 600 Electroporation System, BTX,Genetronics, Inc.). Optimal conditions for electroporation areexperimentally determined for the particular host cell type. Voltage,resistance, and pulse length are the typical parameters varied.Guidelines for optimizing electroporation may be obtained frommanufacturers or found in protocol manuals, such as Current Protocols inMolecular Biology (Ausubel et al. (ed.), Wiley Interscience, 1987).Cells are diluted with an equal volume of medium and incubated for 48hours. Electroporation may be performed on various cell types, includingmammalian cells, yeast cells, bacteria, and the like. Followingincubation, medium with or without inhibitor is added and cells arefurther incubated for 1–2 days. At this time, culture medium isharvested and the protein is assayed by any of the assays herein.Preferably, ELISA is employed to detect the protein. An initialconcentration of 50 μM is tested. If this amount gives a statisticallysignificant reduction of export or reduction of monoclonal Ab detection,the candidate inhibitor is further tested in a dose response.

Alternatively, concentrated supernatant may be electrophoresed on anSDS-PAGE gel and transferred to a solid support, such as nylon ornitrocellulose. MIF is then detected by an immunoblot (see Harlow,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988),using an antibody to MIF containing an isotopic or non-isotopic reportergroup. These reporter groups include, but are not limited to enzymes,cofactors, dyes, radioisotopes, luminescent molecules, fluorescentmolecules, and biotin. Preferably, the reporter group is ¹²⁵I orhorseradish peroxidase, which may be detected by incubation with2,2′-azino-di-3-ethylbenzthiazoline sulfonic acid. These detectionassays described above are readily adapted for use if MIF contains apeptide tag. In such case, the antibody binds to the peptide tag. Otherassays include size or charge-based chromatography, including HPLC, andaffinity chromatography.

Alternatively, a bioassay may be employed to quantify the amount ofactive MIF present in the cell medium. For example, the bioactivity ofthe MIF may be measured by a macrophage migration assay. Briefly, cellstransfected with an expression vector containing MIF are cultured forapproximately 30 hours, during which time a candidate inhibitor orenhancer is added. Following incubation, cells are transferred to a lowserum medium for a further 16 hours of incubation. The medium is removedand clarified by centrifugation. A lysis buffer containing proteaseinhibitors is added to the medium or, in the alternative, cells arelysed and internal levels are determined as follows. Bioactivity of MIFis then measured by macrophage migration assay, isomerase activity, or aproliferation assay. A proliferation assay is performed by addingvarious amounts of the eluate to cultured quiescent 3T3 cells. Tritiatedthymidine is added to the medium and TCA precipitable counts aremeasured approximately 24 hours later. Reduction of the vital dye MTT isan alternative way to measure proliferation. For a standard, purifiedrecombinant human FGF-2 (fibroblast growth factor 2) may be employed.Other functions may be assayed in other appropriate bioassays availablein the art, such as capsular polysaccharides (CPS) induced toxic shock,TSST-1 induced toxic shock, collagen induced arthritis, and the like.

Other in vitro angiogenic assays include bioassays that measureproliferation of endothelial cells within collagen gel (Goto et al., LabInvest. 69:508, 1993), co-culture of brain capillary endothelial cellson collagen gels separated by a chamber from cells exporting the MIFprotein (Okamure et al., B.B.R.C. 186:1471, 1992; Abe et al., J. Clin.Invest. 92:54, 1993), or a cell migration assay (see Warren et al., J.Clin. Invest. 95:1789, 1995).

Production of Antibodies

The term “antibody,” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to polyclonal,monospecific, and monoclonal antibodies, as well as antigen bindingfragments of such antibodies. With regard to an anti-MIF/target antibodyof preferred embodiments, the term “antigen” as used herein is a broadterm and is used in its ordinary sense, including, without limitation,to refer to a macrophage migration inhibitory factor polypeptide or atarget polypeptide, variant, or functional fragment thereof. Ananti-MIF/target antibody, or antigen binding fragment of such anantibody, may be characterized as having specific binding activity forthe target polypeptide or epitope thereof of at least about 1×10⁵ M⁻¹,generally at least about 1×10⁶ M⁻¹, and preferably at least about 1×10⁸M⁻¹. Fab, F(ab′)₂, Fd and Fv fragments of an anti-MIF/target antibody,which retain specific binding activity for a MIF/targetpolypeptide-specific epitope, are encompassed within preferredembodiments. Of particular interest are those antibodies that bindactive polypeptides and are displaced upon binding of an inhibitorysmall molecule. Those of skill in the art readily appreciate that suchdisplacement can be the result of a conformational change, thus changingthe nature of the epitope, competitive binding with the epitope, orsteric exclusion of the antibody from its epitope. In one example, theactive site of an enzyme may be the epitope for a particular antibodyand upon binding of a small molecule at or near the active site,immunoreactivity of the antibody is lost, thereby allowing the use ofloss of immunoreactivity with an antibody as a surrogate marker forenzyme activity.

In addition, the term “antibody” as used herein is a broad term and isused in its ordinary sense, including, without limitation, to refer tonaturally occurring antibodies as well as non-naturally occurringantibodies, including, for example, single chain antibodies, chimeric,bifunctional and humanized antibodies, as well as antigen-bindingfragments thereof. Such non-naturally occurring antibodies may beconstructed using solid phase peptide synthesis, may be producedrecombinantly, or may be obtained, for example, by screeningcombinatorial libraries including variable heavy chains and variablelight chains (Huse et al., Science 246:1275–1281 (1989)). These andother methods of making, for example, chimeric, humanized, CDR-grafted,single chain, and bifunctional antibodies are well known in the art(Winter and Harris, Immunol. Today 14:243–246 (1993); Ward et al.,Nature 341:544–546 (1989); Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, New York (1992); Borrabeck,Antibody Engineering, 2d ed., Oxford Univ. Press (1995); Hilyard et al.,Protein Engineering. A practical approach, IRL Press (1992)).

In certain preferred embodiments, an anti-MIF/target antibody may beraised using as an immunogen such as, for example, an isolated peptideincluding the active site region of MIF or the target polypeptide, whichcan be prepared from natural sources or produced recombinantly, asdescribed above, or an immunogenic fragment of a MIF/target polypeptide(e.g., immunogenic sequences including 8–30 or more contiguous aminoacid sequences), including synthetic peptides, as described above. Anon-immunogenic peptide portion of a MIF/target polypeptide can be madeimmunogenic by coupling the hapten to a carrier molecule such as bovineserum albumin (BSA) or keyhole limpet hemocyanin (KLH), or by expressingthe peptide portion as a fusion protein. Various other carrier moleculesand methods for coupling a hapten to a carrier molecule are well knownin the art (Harlow and Lane, supra, 1992).

Methods for raising polyclonal antibodies, for example, in a rabbit,goat, mouse, or other mammal, are well known in the art. In addition,monoclonal antibodies may be obtained using methods that are well knownand routine in the art (Harlow and Lane, supra, 1992). For example,spleen cells from a target polypeptide-immunized mammal can be fused toan appropriate myeloma cell line such as SP/02 myeloma cells to producehybridoma cells. Cloned hybridoma cell lines may be screened using alabeled target polypeptide or functional fragment thereof to identifyclones that secrete target polypeptide monoclonal antibodies having thedesired specificity. Hybridomas expressing target polypeptide monoclonalantibodies having a desirable specificity and affinity may be isolatedand utilized as a continuous source of the antibodies, which are useful,for example, for preparing standardized kits. Similarly, a recombinantphage that expresses, for example, a single chain anti-targetpolypeptide also provides a monoclonal antibody that may be employed forpreparing standardized kits.

Applications and Methods Utilizing Inhibitors of MIF

Inhibitors of MIF have a variety of applicable uses, as noted above.Candidate inhibitors of MIF may be isolated or procured from a varietyof sources, such as bacteria, fungi, plants, parasites, libraries ofchemicals (small molecules), peptides or peptide derivatives and thelike. Further, one of skill in the art recognize that inhibition hasoccurred when a statistically significant variation from control levelsis observed.

Given the various roles of MIF in pathology and homeostasis, inhibitionof MIF activity or MIF extracellular localization may have a therapeuticeffect. For example, recent studies have demonstrated that MIF is amediator of endotoxemia, where anti-MIF antibodies fully protected micefrom LPS-induced lethality. See Bernhagen et al., Nature 365:756–759,1993; Calandra et al., J. Exp. Med. 179:1895–1902, 1994; Bernhagen etal., Trends Microbiol. 2:198–201, 1994. Further, anti-MIF antibodieshave markedly increased survival in mice challenged with gram-positivebacteria that induces septic shock. Bernhagen et al., J. Mol. Med.76:151–161, 1998. Other studies have demonstrated the role of MIF intumor cell growth and that anti-sense inhibition of MIF leads toresistance to apoptotic stimuli. Takahashi et al., Mol. Med. 4:707–714,1998; Takahashi et al., Microbiol. Immunol. 43(1):61–67, 1999. Inaddition, the finding that MIF is a counterregulator of glucocorticoidaction indicates that methods of inhibiting MIF extracellularlocalization may allow for treatment of a variety of pathologicalconditions, including autoimmunity, inflammation, endotoxemia, and adultrespiratory distress syndrome, inflammatory bowel disease, otitis media,inflammatory joint disease and Crohn's disease. Bernhagen et al., J.Mol. Med. 76:151–161, 1998; Calandra et al., Nature 377:68–71, 1995;Donnelly et al., Nat. Med. 3:320–323, 1997. Because MIF is alsorecognized to be angiogenic, the inhibition of this cytokine may haveanti-angiogenic activity and particular utility in angiogenic diseasesthat include, but are not limited to, cancer, diabetic retinopathy,psoriasis, angiopathies, fertility, obesity and genetic diseases ofglucocorticoid dysfunction like Cushings and Addisons disease.

The inhibitors of MIF activity or export may be employed therapeuticallyand also utilized in conjunction with a targeting moiety that binds acell surface receptor specific to particular cells. Administration ofinhibitors or enhancers generally follows established protocols.Compositions of preferred embodiments may be formulated for the mannerof administration indicated, including for example, for oral, nasal,venous, intracranial, intraperitoneal, subcutaneous, or intramuscularadministration. Within other embodiments, the compositions describedherein may be administered as part of a sustained release implant.Within yet other embodiments, compositions of preferred embodiments maybe formulized as a lyophilizate, utilizing appropriate excipients thatprovide stability as a lyophilizate, and subsequent to rehydration.

In another embodiment, pharmaceutical compositions containing one ormore inhibitors of MIF are provided. For the purposes of administration,the compounds of preferred embodiments may be formulated aspharmaceutical compositions. Pharmaceutical compositions of preferredembodiments comprise one or more MIF inhibitors of preferred embodimentsand a pharmaceutically acceptable carrier and/or diluent. The inhibitorof MIF is present in the composition in an amount which is effective totreat a particular disorder, that is, in an amount sufficient to achievedecreased MIF levels or activity, symptoms, and/or preferably withacceptable toxicity to the patient. Preferably, the pharmaceuticalcompositions of preferred embodiments may include an inhibitor of MIF inan amount from less than about 0.01 mg to more than about 1000 mg perdosage depending upon the route of administration, preferably about0.025, 0.05, 0.075, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 mg toabout 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250,300, 350, 375, 400, 425, 450, 500, 600, 700, 800, or 900 mg, and morepreferably from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg toabout 30, 35, 40, 45, 50, 55, or 60 mg. In certain embodiments, however,lower or higher dosages than those mentioned above may be preferred.Appropriate concentrations and dosages can be readily determined by oneskilled in the art.

Pharmaceutically acceptable carriers and/or diluents are familiar tothose skilled in the art. For compositions formulated as liquidsolutions, acceptable carriers and/or diluents include saline andsterile water, and may optionally include antioxidants, buffers,bacteriostats, and other common additives. The compositions can also beformulated as pills, capsules, granules, or tablets that contain, inaddition to an inhibitor of MIF, diluents, dispersing and surface-activeagents, binders, and lubricants. One skilled in this art may furtherformulate the inhibitor of MIF in an appropriate manner, and inaccordance with accepted practices, such as those described inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990.

In addition, prodrugs are also included within the context of preferredembodiments. Prodrugs are any covalently bonded carriers that release acompound of structure (Ia) or (Ib) in vivo when such prodrug isadministered to a patient. Prodrugs are generally prepared by modifyingfunctional groups in a way such that the modification is cleaved, eitherby routine manipulation or in vivo, yielding the parent compound.

With regard to stereoisomers, the compounds of structures (Ia) and (Ib)may have chiral centers and may occur as racemates, racemic mixtures andas individual enantiomers or diastereomers. All such isomeric forms areincluded within preferred embodiments, including mixtures thereof.Furthermore, some of the crystalline forms of the compounds ofstructures (Ia) and (Ib) may exist as polymorphs, which are included inpreferred embodiments. In addition, some of the compounds of structures(Ia) and (Ib) may also form solvates with water or other organicsolvents. Such solvates are similarly included within the scope of thepreferred embodiments.

In another embodiment, a method is provided for treating a variety ofdisorders or illnesses, including inflammatory diseases, arthritis,immune-related disorders, and the like. Such methods includeadministering of a compound of preferred embodiments to a warm-bloodedanimal in an amount sufficient to treat the disorder or illness. Suchmethods include systemic administration of an inhibitor of MIF ofpreferred embodiments, preferably in the form of a pharmaceuticalcomposition. As used herein, systemic administration includes oral andparenteral methods of administration. For oral administration, suitablepharmaceutical compositions of an inhibitor of MIF include powders,granules, pills, tablets, and capsules as well as liquids, syrups,suspensions, and emulsions. These compositions may also includeflavorants, preservatives, suspending, thickening and emulsifyingagents, and other pharmaceutically acceptable additives. For parentaladministration, the compounds of preferred embodiments can be preparedin aqueous injection solutions that may contain, in addition to theinhibitor of MIF activity and/or export, buffers, antioxidants,bacteriostats, and other additives commonly employed in such solutions.

As mentioned above, administration of a compound of preferredembodiments can be employed to treat a wide variety of disorders orillnesses. In particular, the compounds of preferred embodiments may beadministered to a warm-blooded animal for the treatment of inflammation,cancer, immune disorders, and the like.

MIF inhibiting compounds may be used in combination therapies with otherpharmaceutical compounds. In preferred embodiments, the MIF inhibitingcompound is present in combination with conventional drugs used to treatdiseases or conditions wherein MIF is pathogenic or wherein MIF plays apivotal or other role in the disease process. In particularly preferredembodiments, pharmaceutical compositions are provided comprising one ormore MIF inhibiting compounds, including, but not limited to compoundsof structures (1a) or (1b), in combination with one or more additionalpharmaceutical compounds, including, but not limited to drugs for thetreatment of various cancers, asthma or other respiratory diseases,sepsis, arthritis, inflammatory bowel disease (IBD), or otherinflammatory diseases, immune disorders, or other diseases or disorderswherein MIF is pathogenic.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with one or more nonsteroidalanti-inflammatory drugs (NSAIDs) or other pharmaceutical compounds fortreating arthritis or other inflammatory diseases. Preferred compoundsinclude, but are not limited to, celecoxib; rofecoxib; NSAIDS, forexample, aspirin, celecoxib, choline magnesium trisalicylate, diclofenacpotassium, diclofenac sodium, diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, melenamicacid, nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam,rofecoxib, salsalate, sulindac, and tolmetin; and corticosteroids, forexample, cortisone, hydrocortisone, methylprednisolone, prednisone,prednisolone, betamethesone, beclomethasone dipropionate, budesonide,dexamethasone sodium phosphate, flunisolide, fluticasone propionate,triamcinolone acetonide, betamethasone, fluocinolone, fluocinonide,betamethasone dipropionate, betamethasone valerate, desonide,desoximetasone, fluocinolone, triamcinolone, triamcinolone acetonide,clobetasol propionate, and dexamethasone.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with one or more beta stimulants,inhalation corticosteroids, antihistamines, hormones, or otherpharmaceutical compounds for treating asthma, acute respiratorydistress, or other respiratory diseases. Preferred compounds include,but are not limited to, beta stimulants, for example, commonlyprescribed bronchodilators; inhalation corticosteroids, for example,beclomethasone, fluticasone, triamcinolone, mometasone, and forms ofprednisone such as prednisone, prednisolone, and methylprednisolone;antihistamines, for example, azatadine, carbinoxamine/pseudoephedrine,cetirizine, cyproheptadine, dexchlorpheniramine, fexofenadine,loratadine, promethazine, tripelennamine, brompheniramine,cholopheniramine, clemastine, diphenhydramine; and hormones, forexample, epinephrine.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with pharmaceutical compounds fortreating IBD, such as azathioprine or corticosteroids, in apharmaceutical composition.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with pharmaceutical compounds fortreating cancer, such as paclitaxel, in a pharmaceutical composition.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with immunosuppresive compounds ina pharmaceutical composition. In particularly preferred embodiments, oneor more MIF inhibiting compounds are present in combination with one ormore drugs for treating an autoimmune disorder, for example, Lymedisease, Lupus (e.g., Systemic Lupus Erythematosus (SLE)), or AcquiredImmune Deficiency Syndrome (AIDS). Such drugs may include proteaseinhibitors, for example, indinavir, amprenavir, saquinavir, lopinavir,ritonavir, and nelfinavir; nucleoside reverse transcriptase inhibitors,for example, zidovudine, abacavir, lamivudine, idanosine, zalcitabine,and stavudine; nucleotide reverse transcriptase inhibitors, for example,tenofovir disoproxil fumarate; non nucleoside reverse transcriptaseinhibitors, for example, delavirdine, efavirenz, and nevirapine;biological response modifiers, for example, etanercept, infliximab, andother compounds that inhibit or interfere with tumor necrosing factor;antivirals, for example, amivudine and zidovudine.

In particularly preferred embodiments, one or more MIF inhibitingcompounds are present in combination with pharmaceutical compounds fortreating sepsis, such as steroids or anti-infective agents. Examples ofsteroids include corticosteroids, for example, cortisone,hydrocortisone, methylprednisolone, prednisone, prednisolone,betamethesone, beclomethasone dipropionate, budesonide, dexamethasonesodium phosphate, flunisolide, fluticasone propionate, triamcinoloneacetonide, betamethasone, fluocinolone, fluocinonide, betamethasonedipropionate, betamethasone valerate, desonide, desoximetasone,fluocinolone, triamcinolone, triamcinolone acetonide, clobetasolpropionate, and dexamethasone. Examples of anti-infective agents includeanthelmintics (mebendazole), antibiotics including aminoclycosides(gentamicin, neomycin, tobramycin), antifungal antibiotics (amphotericinb, fluconazole, griseofulvin, itraconazole, ketoconazole, nystatin,micatin, tolnaftate), cephalosporins (cefaclor, cefazolin, cefotaxime,ceftazidime, ceftriaxone, cefuroxime, cephalexin), beta-lactamantibiotics (cefotetan, meropenem), chloramphenicol, macrolides(azithromycin, clarithromycin, erythromycin), penicillins (penicillin Gsodium salt, amoxicillin, ampicillin, dicloxacillin, nafcillin,piperacillin, ticarcillin), tetracyclines (doxycycline, minocycline,tetracycline), bacitracin; clindamycin; colistimethate sodium; polymyxinb sulfate; vancomycin; antivirals including acyclovir, amantadine,didanosine, efavirenz, foscamet, ganciclovir, indinavir, lamivudine,nelfinavir, ritonavir, saquinavir, stavudine, valacyclovir,valganciclovir, zidovudine; quinolones (ciprofloxacin, levofloxacin);sulfonamides (sulfadiazine, sulfisoxazole); sulfones (dapsone);furazolidone; metronidazole; pentamidine; sulfanilamidum crystallinum;gatifloxacin; and sulfamethoxazole/trimethoprim.

In the treatment of certain diseases, it may be beneficial to treat thepatient with a MIF inhibitor in combination with an anesthetic, forexample, ethanol, bupivacaine, chloroprocaine, levobupivacaine,lidocaine, mepivacaine, procaine, ropivacaine, tetracaine, desflurane,isoflurane, ketamine, propofol, sevoflurane, codeine, fentanyl,hydromorphone, marcaine, meperidine, methadone, morphine, oxycodone,remifentanil, sufentanil, butorphanol, nalbuphine, tramadol, benzocaine,dibucaine, ethyl chloride, xylocalne, and phenazopyridine.

EXAMPLES

The inhibitors of MIF of preferred embodiments may be prepared andscreened for inhibition of activity or export as described in thefollowing examples.

Example 1

Macrophage Migration Assay

Macrophage migration is measured by using the agarose droplet assay andcapillary method as described by Harrington and Stastny et al., J.Immunol. 110(3):752–759, 1973. Briefly, macrophage-containing samplesare added to hematocrit tubes, 75 mm long with a 1.2 mm inner diameter.The tubes are heat sealed and centrifuged at 100×G for 3 minutes, cut atthe cell-fluid interface and imbedded in a drop of silicone grease inSykes-Moore culture chambers. The culture chambers contain either acontrol protein (BSA) or samples. Migration areas are determined after24 and 48 hours of incubation at 37° C. by tracing a projected image ofthe macrophage fans and measuring the areas of the migration byplanimetry.

Alternatively, each well of a 96-well plate is pre-coated with onemicroliter of liquid 0.8% (w/v) Sea Plaque Agarose in water dispensedonto the middle of each well. The plate is then warmed gently on a lightbox until the agarose drops are just dry. Two microliters of macrophagecontaining cell suspensions of up to 25% (v/v) in media (with or withoutMIF or other controls), containing 0.2% agarose (w/v) and heated to 37°C. is added to the precoated plate wells and cooled to 4° C. for 5 min.Each well is then filled with media and incubated at 37° C. under 5% CO₂−95% air for 48 hr. Migration from the agarose droplets is measured at24 and 48 hr by determining the distance from the edge of the droplet tothe periphery of migration.

Migration Assay

Monocyte migration inhibitory activities of recombinant murine and humanwild-type and murine mutant MIF are analyzed by use of human peripheralblood mononuclear cells or T-cell depleted mononuclear cells in amodified Boyden chamber format. Calcein AM-labeled monocytes aresuspended at 2.5 to 5×10⁶/mL in RPMI 1640 medium (medium for the growthof human leukemia cells in monolayer or suspension cultures, fromRoswell Park Memorial Institute), with L-glutamine (without phenol red)and 0.1 mg/mL human serum albumin or bovine serum albumin. An aliquot(200 μL) of cell suspension is added to wells of a U-bottom 96-wellculture plate (Costar, Cambridge, Mass.) prewarmed to 37° C. MIF in RPMI1640 is added to the cell suspension to yield final concentrations of 1,10, 100, and 1000 ng/mL. The culture plate is placed into the chamber ofa temperature-controlled plate reader, mixed for 30 s, and incubated at37° C. for 10–20 min. During the incubation, 28 μL of prewarmed humanmonocyte chemotactic protein 1 (MCP-1; Pepro Tech., Inc., Rocky Hill,N.J.) at 10 or 25 ng/mL or RPMI 1640 with 0.1 mg/mL HSA is added to thebottom well of a ChemoTX plate (Neuro Probe Inc., Gaithersburg, Md.; 3mm well diameter, 5 μM filter pore size). The filter plate is carefullyadded to the base plate. Treated cell suspensions are removed from theincubator and 30 μL is added to each well of the filter plate. Theassembled plate is incubated for 90 min. at 37° C. in a humidifiedchamber with 5% CO₂. Following incubation, the cell suspension isaspirated from the surface of the filter and the filter is subsequentlyremoved from the base plate and washed three times by adding 50 μL of1×HBSS⁻ (Hanks' Balance Salt Solution in the 1×concentration) to eachfilter segment. Between washes, a squeegee (NeuroProbe) is employed toremove residual HBSS⁻. The filter is air-dried and then read directly inthe fluorescent plate reader, with excitation at 485 nm and emission at535 nm. Chemotactic or random migration indices are defined as averagefilter-bound fluorescence for a given set of wells divided by averagefluorescence of filters in wells containing neither MCP-1 nor MIF.Titration of fluorescently labeled cells revealed that levels offluorescence detected in this assay have a linear relationship to cellnumber (not shown).

Tautomerase Assay

The tautomerization reaction is carried out essentially as described byRosengren et al., Mol. Med. 2(1):143–149, 1996. D-dopachrome conversionto 5,6-dihydroxyindole-2-carboxylic acid is assessed. 1 ml samplecuvettes containing 0.42 mM substrate and 1.4 μg of MIF in a samplesolution containing 0.1 mM EDTA and 10 mM sodium phosphate buffer, pH6.0 are prepared and the rate of decrease in iminochrome absorbance isfollowed at 475 nm. L-dopachrome is employed as a control. In addition,the reaction products can be followed using an HPLC, utilizing a mobilephase including 20 mM KH₂PO₄ buffer (pH 4.0) and 15% methanol with aflow rate of 1.2 ml/min. Fluorimetric detection is followed at 295/345nm.

Alternatively, the tautomerization reaction utilizing phenylpyruvate or(p-hydroxyphenyl)pyruvate is carried out essentially as described byJohnson et al., Biochem. 38:16024–16033, 1999. In this version,ketonization of phenylpyruvate is monitored at 288 nm (ε=17300 M⁻¹ cm⁻¹)and the ketonization of (p-hydroxyphenyl)pyruvate is monitored at 300 nm(ε=21600 M⁻¹ cm⁻¹). The assay mixture contains 50 mM Na₂HPO₄ buffer (1mL, pH 6.5) and an aliquot of a solution of MIF sufficiently dilute(0.5–1.0 μL of a 2.3 mg/mL solution, final concentration of 93–186 nM)to yield an initial liner rate. The assay is initiated by the additionof a small quantity (1–3.3 μL) of either phenylpyruvate or(p-hydroxyphenyl)pyruvate from stock solutions made up in ethanol. Thecrystalline forms of phenylpyruvate and (p-hydroxyphenyl)pyruvate existexclusively as the enol isomers (Larsen et al., Acta Chem. Scand. B28:92–96, 1974). The concentration of substrate may range from 10 to 150M, with no significant inhibition of MIF activity by ethanol observed atless than 0.5% v/v.

Immunoprecipitation and Western Blot Analysis

Cell culture experiments are designed to characterize the activity ofcandidate compounds, MIF expression, trafficking, and export. Cell andconditioned medium fractions are prepared for immunoprecipitationessentially as described previously (Florkiewicz et al., Growth Factors4:265–275, 1991; Florkiewicz et al., Ann. N.Y. Acad. Sci. 638:109–126)except that 400 μl of lysis buffer (1% NP-40, 0.5% deoxycholate, 20 mMTris pH 7.5, 5 mM EDTA, 2 mM EGTA, 0.01 mM phenylmethylsufonyl fluoride,10 ng/ml aprotinin, 10 ng/ml leupeptin, 10 ng/ml peptstatin) is added tothe medium fraction after clarification by centrifugation in a microfugefor 15 minutes. Cell or medium fractions are incubated with monoclonalor polyclonal antibodies to MIF and GammaBind™ G Sepharose® (PharmaciaLKB Biotechnology, Uppsala, Sweden) was added for an additional 30minutes incubation. Immune complexes are sedimented by microfugecentrifugation, washed three times with lysis buffer, and four timeswith ice cold immunoprecipitation wash buffer (0.15M NaCl, 0,01 MNa-phosphate pH 7.2, 1% deoxycholate, 1% NP-40, 0.1% sodium dodecylsulfate). Immune complexes are dissociated directly in SDS gel samplebuffer 125 mM Tris, pH 6.8, 4% SDS, 10% glycerol, 0.004% bromphenolblue, 2 mM EGTA, and separated by 12% SDS-PAGE. The gel is processed forfluorography, dried, and exposed to X-ray film at −70° C. When neomycinphosphotransferase is immunoprecipitated, a rabbit anti-NPT antibody(5Prime-3Prime, Boulder, Colo.) was employed.

For Western blot analysis, proteins are transferred from the 12%SDS-PAGE gel to a nitrocellulose membrane (pore size 0.45 μm in coldbuffer containing 25 mM3-[dimethyl(hydroxymethyl)methylamino]-2-hydroxypropane-sulfonic acid,pH 9.5, 20% methanol for 90 minutes at 0.4 amps. For Western blottinganalysis, of cell conditioned media, the media was centrifuged (10minutes at 800 g) and the supernatants concentrated 10-fold by membranefiltration (10 kDa cut-off, Centricon-10 Amicon). Samples were thenresolved on 16% SDS Tris-glycin Gel (Novex, San Diego, Calif.) underreducing condition and transferred onto nitrocellulose membrane (Novex)at 20V for 3 hours. Membrane was incubated with rabbit polyclonalanti-rat antibodies (1:1000) (Torrey Pines Biolab, San Diego, Calif.),and then with horseradish peroxidase-conjugate (1:1000)(Pierce,Rockford, Ill.). MIF was visualized by development withchloronaphtnol/H₂O₂. Recombinant MIF (2 ng, purchased from R&D systems,Minneapolis) was electrophoresed and transferred as a standard.Membranes are blocked in 10 mM Tris, pH 7.5, 150 mM NaCl, 5 mM NaN₃,0.35% polyoxyethylene-sorbitan monolaurate, and 5% nonfat dry milk(Carnation Co., Los Angeles, Calif.) for 1 hr at room temperature.Membranes are incubated with a monoclonal antibody (Catalog NumberMAB289, purchased from R&D Systems, Minneapolis, Minn.) or polyclonal(goat polyclonal serum, R&D Systems cat#AF-289-PB). Followingincubation, membranes are washed at room temperature with 10 changes ofbuffer containing 150 mM NaCl, 500 mM sodium phosphate pH 7.4, 5 mMNaN₃, and 0.05% polyoxyethylene-sorbitan monolaurate. When usingmonoclonal antibodies, membranes are then incubated in blocking buffercontaining 1 μg/ml rabbit anti-mouse IgG (H+L, affinipure, JacksonImmuno Research Laboratories, West Grove, Pa.) for 30 minutes at roomtemperature. For polyclonal probing, incubation employed rabbitanti-goat (Sigma, Catalog Number G5518). Membranes are subsequentlywashed in 1 L of buffer described above, and incubated for 1 hr in 100ml of blocking buffer containing 15 μCi ¹²⁵I-protein A (ICNBiochemicals, Costa Mesa, Calif.), and washed with 1 L of buffer. Theradiosignal is visualized by autoradiography.

Overnight conditioned media is collected from LPS (10 μg/ml) treatedTHP-1 cells also treated with varying amounts of candidate compounds andscreened by immunoprecipitation with monoclonal or polyclonal antibodiesto detect MIF binding. Conditioned media show a significant loss ofdetectable MIF using the monoclonal antibody in the presence of 10 μM ofcandidate compounds that is not observed with the polyclonal antibody.This response mirrors the effect of candidate compounds on MIF enzymeactivity. Accordingly, monoclonal reactivity acts as a surrogate markerfor enzymatic activity.

Varying concentrations of inhibitor analogs are added to LPS stimulatedTHP-1 cells and allowed to incubate overnight. The following day theamount of immunoreactive MIF detected is evaluated by ELISA. Candidatecompounds inhibit the ability of the antibody to recognize MIF.

The ability of candidate compounds to decrease the immunoreactivity ofMIF produced by THP-1 cells is determined. THP-1 cells are treated with10 μg/ml of LPS and 10 μM of the candidate compound is added at varioustimes post-LPS stimulation and immunoreactivity monitored with ananti-MIF monoclonal. Following addition of candidate compounds,immunoreactivity is rapidly lost. The activity of compounds or bufferalone controls on MIF detection is measured when the candidate compoundsare initially added at various times to cell cultures and then thecorresponding conditioned media samples are processed in a timedependent fashion.

The ability of candidate compounds to modulate antibody recognition ofMIF is examined using pre-conditioned media, in the absence of livecells. In this experiment, LPS is added to THP1 cells in culture asdescribe above. Six hours later, the conditioned media is removed,clarified of cell debris and the amount of MIF determined to be 22ng/ml. This pre-conditioned media is then divided into two groups. Bothgroups are incubated at 37° C. for varying periods of time before acandidate compound or buffer alone (control) is added for an additional30 minutes of incubation at 37° C. The level of detectable MIF is thendetermined by ELISA using the monoclonal anti-MIF antibody fordetection. The rapid loss of MIF specific ELISA signal is dependent uponthe presence of the candidate compound. Control levels of MIF do notchange. Accordingly, candidate compounds interact with MIF, and blockthe antibody's ability to subsequently interact with MIF, even in theabsence of cells. As this interaction takes place at the catalytic site,or constrains catalytic activity, the loss of immunoreactivitycorrelates with lost enzymatic activity and/or MIF associatedactivities.

Extracellular Localization Assay

In order to further assess in vitro activity of candidate compounds tomodulate MIF export, mouse macrophage RAW 264.7 cells (a murinemacrophage cell line, American Type Culture Collection, Manassas, Va.)are selected.

Raw 264.7 macrophage (3×10⁶ cells per well) are plated in 12-well tissueculture plates (Costar) and cultured in RPMI/1% heat-inactivated fetalbovine serum (FBS) (Hyclone Laboratories, Logan, Utah). After threehours of incubation at 37° C. in a humidified atmosphere with 5% CO₂,nonadherent cells are removed and wells are washed twice with RPMI/1%FBS. Cells are then incubated for 24 hours with LPS (0111:B4) or TSST-1(Toxic Shock Syndrome Toxin-1, Toxin Technology, Sarasota, Fla.), whichare approximately 95% pure and resuspended in pyrogen-free water, at aconcentration ranging from 1 pg/ml to 1000 ng/ml (for the dose responseexperiment). For time-course experiments, conditioned media of parallelcultures are removed at 0.5, 1, 2, 4, 8 and 24 hours intervals afterstimulation with 1 ng/ml TSST-1 or LPS. For the inhibition studies, RAW264.7 cells (3×10⁶ cells per well) are incubated for 24 hours with 1ng/ml of LPS (0111:B4) or 1 ng/ml of TSST-1 in the presence of 0.01 μMto 10 μM candidate compound or buffer (as control). The MIF incell-conditioned media is concentrated on filters and the MIF remainingin the samples is analyzed by Western blotting and MIF band densitiesare also measured by Stratagene Eagle Eye™.

RAW cells can be induced to express MIF by addition of either 1 ng/mlTSST-1 or LPS and cultured for 24 hours. MIF in conditioned media ismeasured as described above. Candidate compounds reduce immunodetectableMIF levels in conditioned media in a concentration dependent manner, ascompared to cells incubated with buffer only.

Cell Culture, Transfection, and Metabolic Labeling

Target cells obtained from the American Type Culture Collection (ATCCNo. CRL 1650) are cultured overnight in a 48-well plate in DMEM(Dulbecco's Modified Eagles Medium) supplemented with 10% fetal bovineserum, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 nM nonessential aminoacids, and 50 μg/ml gentamycin. The target cells are then transfectedwith 2 μg/ml of CsCl-purified plasmid DNA in transfection buffer (140 mMNaCl, 3 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 0.9 mM Na₂HPO₄, 25 mM Tris, pH7.4. To each well, 300 μl of the DNA in transfection buffer is added.Cells are incubated for 30 minutes at 37° C., and the buffer isaspirated. Warm medium supplemented with 100 μm chloroquine is added for1.5 hr. This medium is removed and the cells are washed twice withcomplete medium. Cells are then incubated for 40–48 hr. The plasmid ofinterest is co-transfected with pMAMneo (glucocorticoid-induciblemammalian expression vector containing a neomycin-resistant gene,Clontech, Palo Alto, Calif.), which contains the selectable markerneomycin phosphotransferase. When 2 μg of the plasmid of interest areco-transfected with 10 μg of pMAMneo, greater than 70% of transfectedcells express both MIF and neo, as determined by immunofluorescencemicroscopy.

For immunoprecipitation assays the target cells are metabolicallypulse-labeled for 15 minutes with 100 μCi of ³⁵S-methionine and³⁵S-cysteine (Trans ³⁵S-label, ICN Biomedicals, Irvine, Calif.) in 1 mlof methionine and cysteine free DMEM. Following labeling, the cellmonolayers are washed once with DMEM supplemented with excess (10 mM)unlabeled methionine and cysteine for 1–2 minutes. Cells are thencultured in 2 ml of this medium for the indicated lengths of time andthe cell supernatants are immunoprecipitated for the presence ofleaderless protein. For the indicated cultures, chase medium issupplemented with modulator at the indicated concentrations.

Alternatively, for analysis by ELISA, the target cells are washed oncewith 250 μl of 0.1 M sodium carbonate, pH 11.4, for 1 to 2 minutes andimmediately aspirated. A high salt solution may alternatively bepreferred. The cells are washed with media containing 0.5% FBS plus 25μg/ml heparin and then the cells are incubated in this same medium forthe indicated lengths of time. For indicated cultures, chase medium issupplemented with a modulator. For cells transfected with vectorencoding a protein containing a leader sequence, such as hCG-α or anyother non-heparin binding protein, the carbonate wash and heparincontaining medium may be omitted.

High Throughput Screening Assay for MIF Inhibitors

The high throughput screening assay for MIF inhibitors is performed in a96-well format using MIF produced by THP-1 cells and is performed asfollows. MIF assays are performed by ELISA as indicated above. THP-1cells are resuspended to approx. 5×10⁶ cells/ml in RPMI mediumcontaining 20 μg/ml of bacterial LPS and the cells incubated for 18–20hours. Subsequently cell supernatant is collected and incubated withputative inhibitors. Briefly, a 96-well plate (Costar Number 3590) ELISAplate is coated with a MIF monoclonal antibody (R&D Systems CatalogNumber MAB289) at a concentration of 4 μg/ml for two hours at 37° C.Undiluted culture supernate is added to the ELISA plate for a two-hourincubation at room temperature. The wells are then washed, abiotinylated MIF polyclonal antibody (R&D Systems #AF-289-PB) is addedfollowed by Streptavidin-HRP and a chromogenic substrate. The amount ofMIF is calculated by interpolation from an MIF standard curve.

HPLC Analysis of Candidate Inhibitors in Serum

Prior to evaluating the affects of any small molecule in vivo, it isdesirable to be able to detect, in a quantitative fashion, the compoundin a body fluid such as blood. An analytical method was established tofirst reproducibly detect test compounds, such as MIF inhibitors, andthen measure its concentration in biological fluid.

RP-HPLC is performed with a Hewlett-Packard Model HP-1100 unit usingSymmetry Shield RP-8 (4.6×75 mm id, Waters, Milford, Mass.). The mobilephase is an isocratic solution of 35% Acetonitrile/water containing 0.1%trifluroacetic acid. Absorbance is monitored at 235 nm. To measure theamount of test compound in serum, the sample serum proteins are firstseparated using 50% Acetonitrile (4° C. overnight) followed bycentrifugation at 14000 rpm for 30 minutes. The supernatant is thenanalyzed by the RP-HPLC and the compound concentration calculated basedon a calibration curve of known standard. According to this procedure,reverse phase HPLC is employed to detect a candidate compound in alinear range of 1.5–800 ng (R2=1) using a spiked test samples (notshown). When the above analytical technique is applied to blood serumfrom animals receiving candidate compounds, circulating concentrationsof candidate compounds are quantitatively measured.

With the development of the above methods, it is possible to evaluatethe efficacy of different routes of compound administration and tocharacterize bioactivity. To test time dependent serum bioavailability,animals are treated with candidate compounds by intraperitonealinjection (i.p.), and orally by gavage.

In Vivo Inhibition of MIF

The purpose for the following in vivo experiments is to confirm initialin vitro assay results using candidate compounds to inhibit MIF.LPS-induced toxicity appears to be related to an overproduction of MIFas well as TNF-α and IL-1β. Since animals can be protected fromendotoxin shock by neutralizing or inhibiting these inflammationmediators. The present model was chosen because it provides reproducibleand rapid lethal models of sepsis and septic shock.

Doses of lipopolysacchraride (LPS) are made fresh prior to eachexperiment. LPS (Escherichia coli 0111:B4, Sigma) is reconstituted byadding 0.5% TEA (1 ml USP water+5 ml Triethylamine (Pierce)) to a vialof 5 mg endotoxin. Once reconstituted, the solution is incubated at 37°C. for 30 minutes. Subsequently, the solution is sonicated in a 56–60°C. bath sonicator for 30 seconds 3 times. Following sonication themixture is vortexed for 3 minutes continuously. The stock solution ofLPS is then ready for use.

Detection of IL-1β and TNF-α and MIF in Blood

Ten 10-week-old (20±2 gram) female BALB/c mice (Charles RiverLaboratories, Kingston, N.Y.) are housed in a group of five per cagewith free access to food and water and are acclimatized for at least oneweek prior to experimentation. On the day of experiment, mice areweighed and randomly distributed into groups of ten animals of equalmean body weight. Mice are injected i.p. with 200 μL of formulatedcandidate compounds or buffer alone immediately before the i.p.injection of LPS (Escherichia coli 0111:B4, 10 mg/kg or 5 mg/kg bodyweight) and β-D-galactosamine (50 mg/kg body weight). Each dose of LPS(0.2 ml for 20 gram mouse) is administered intraperitoneally and mixedwith a final concentration of β-D-galactosamine of 50 mg per ml.Following collection of blood specimens taken from cardiac puncture, theanimal is sacrificed. Typical collections are performed at 4 hours postLPS treatment. The serum is separated in a serum separator (Microtainer®Becton Dickinson, Minneapolis, N.J.) according to the manufacturer'sprotocol. Mouse serum Il-1β and TNF-α are measured by ELISA using a“mouse IL 1β immunoassay or mouse TNF-α immunoassay” kits (R&D SystemMinneapolis, Minn.) following manufacturer's direction. Serum MIFconcentrations in mouse serum are quantified by a sandwich ELISA(ChemiKine MIF Kit, Chemicon, San Diego, Calif.). Samples are analyzedin duplicate, and results are averaged.

Murine LPS Model

Ten 8 to 10 week-old (20±2 gram) female BALB/c mice are housed andacclimatized as described above. On the day of the experiments, the miceare weighed and randomly distributed into groups of five animals ofequal mean body weight. Mice are injected with 200 μl of formulatedcandidate compounds or their Buffer (average 20 mg/kg compound)following i.p. injection of LPS (E. coli 055B5, Sigma) (40, 10, 5, 2 or0.5 mg/kg body weight) and 50 mg/kg of β-D-galactosamine. Mice areobserved every two hours during the first 18 hours and twice a day forseven days. For these studies Kaplan-Meier estimation methods areemployed to assess animal survival.

For all in vivo studies, standard statistical comparisons amongtreatment groups are performed using the Fisher test for categoricaldata and the Mantel-Cox test for continuous variables. To determine iflevels of serum IL-1 correlated to serum MIF, a Fisher's test isapplied. The analyses are performed using Stat View 5.0 Software (AbacusConcepts, Berkeley, Calif.). All reported p values that are two-sidedand of a value less than 0.05 are considered to indicate statisticalsignificance.

An initial control experiment is conducted to determine the base linelevels of endogenous MIF in the murine model system (female Balb/cmice), and further to determine the rate and extent of increase inendogenous MIF following treatment with LPS (10 mg/kg). Female Balb/cmice are treated with LPS (Sigma 0111:B1) admixed with 50 mg/kgβ-D-galactosamine. The level of MIF in serum is measured by HPLC asdescribed above at 0, 2, 5 and 6 hours following LPS/galactosaminetreatment. At the initiation of this representative experiment, thebaseline level of endogenous MIF is determined When mice are treatedwith candidate compounds (formulated in 50% aqueous solution) and 10mg/kg of LPS there is a significant decrease in the level of circulatingMIF that can be detected. In a further experiment, both MIF and IL-1βare measured in mouse serum via ELISA. A direct and highly significantcorrelation between the two is observed in MIF and IL-1β. Thiscorrelation is also observed between MIF and TNF-α. In a similarexperiment, reductions in serum IL-1β level and serum TNF-α level areobserved following administration of 20 mg/kg of candidate compound.

Studies of experimental toxic shock induced by LPS have revealed acentral role for MIF and TNF-α. The fact that LPS stimulatesmacrophage-like cells to produce MIF, that in turn induce TNF-αsecretion by macrophage like cells suggests a potential role for MIF inthe pathogenesis of LPS. To test if candidate compounds can prevent LPSshock, a model of lethal LPS mediated shock in BALB/c mice sensitizedwith β-D-galactosamine is employed. Treatment with candidate compoundsat the time of injection of a lethal dose of LPS (2, 5 and 10 mg/kg)increases survival. The effects are modulated by the concentration ofLPS employed, demonstrating that when using a higher concentration ofLPS, the effect of the candidate compound is saturable and hencespecific.

MIF Overcomes the Effects of Candidate Compounds

Exogenous recombinant human MIF when administered with candidatecompounds can reverse the beneficial effects of the compound, supportingthe hypothesis that candidate compounds act to increase animalresistance to LPS by modulating MIF levels in mice serum. In thisexample, mice are treated with the standard LPS protocol except that inaddition to 1 mg/kg LPS and 20 mg/kg of a candidate compound, someanimals also receive 300 μg/kg human recombinant MIF. At 12 hours,significantly more mice survive the LPS with candidate compounds, butthis survival is neutralized by the administration of MIF.

MIF Inhibitor in a Collagen Induced Arthritis Model

Twenty DBA/1LacJ mice, age 10 to 12 weeks, are immunized on Day 0 atbase of the tail with bovine collagen type II (CII 100 μg) emulsified inFreunds complete adjuvant (FCA; GibcoBRL). On Day 7, a second dose ofcollagen is administrated via the same route (emulsified in Freundsincomplete adjuvant). On Day 14 mice are injected subcutaneously with100 mg of LPS (055:B5). On Day 70 mice are injected 40 μg LPS (0111:B4)intraperitoneally. Groups are divided according paw thickness, which ismeasured by a caliper, after randomization, to create a balancedstarting group. Candidate compound in buffer is given to mice on Days71, 72, 73, and 74 (total eight doses at 0.4 mg/dose, approximately 20mg/kg of body weight). Mice are then examined on Day 74 by two observersfor paw thickness. In this experiment, subsided mice (decline offull-blown arthritis) are treated with a final i.p. injection of LPS onDay 70 to stimulate cytokine production as well as acute inflammation.Candidate compound treated mice develop mildly reduced edema of the pawcompared with vehicle only treated controls. In the late time point, theanimals in the treated group do not reach a full-blown expression ofcollagen induced arthritis as compared to its control.

In another experiment, fifteen DBA/1J mice, age 10 to 12 weeks areimmunized on Day 0 at the base of the tail with bovine collagen type II(CII 100 μg), emulsified in Freunds complete adjuvant (FCA; GibcoBRL).On Day 21, a second dose of collagen is administered via the same route,emulsified in Freunds incomplete adjuvant. On Day 28 the mice areinjected subcutaneously with 100 μg of LPS (055:B5). On Day 71 the miceare injected i.p. with 40 μg LPS (0111:B4). Groups and treatmentprotocol are the same as described as above. On Day 74 blood samples arecollected and cytokines were measured. Candidate compounds reduce serumMIF levels as compared to untreated CIA samples. An even moresignificant inhibition of serum TNF-α levels is detected.

Example 2

Inhibitors of MIF of certain embodiments may be prepared according tothe following reaction schemes. Each of these MIF inhibitors belongs toone of the classes of compounds described above. The variables R₁, R₂,R₃, R₄, Z, and n are as defined above.

General Methods for the Synthesis of the Compounds of the Inventions

The compounds were synthesized starting from substituted orunsubstituted isatoic anhydrides. The strategies to introduce R₂ and/orR₃ groups into the compounds of structures (1a) and (1b) described aboveinvolved preparation of substituted isatoic anhydrides as precursorcompounds from substituted anthranilic acids. The substitutedanthranilic acids were prepared from substituted nitrobenzoic acids. Insome cases, the nitro benzoic acids were obtained by nitration ofappropriate benzoic acid, as shown in Reaction Scheme 1, however, anysuitable method for preparation of nitrobenzoic acids may be employed.

Two different methods were employed to introduce the R₁ group into thecompounds of structures (1a) and (1b) as described above. In one method,the substituted isatoic anhydrides prepared as described in ReactionScheme 1 were alkylated in the N-1 position, then converted to thesubstituted quinolinone intermediate of structure i (depicted inReaction Scheme 2 below). Amination of intermediate i yielded amideintermediate of structure ii which was reacted withphosphorousoxychloride to yield an intermediate of type iii as depictedin Reaction Scheme 2.

To introduce the R₄ group into the compounds of structures (1a) and(1b), the chloro intermediate of structure iii was either reacted withacylated piperazine or reacted first with excess piperazine to yield anintermediate of structure iv, and then acylated to yield the targetcompound as depicted in Reaction Scheme 3. Acylation of intermediate ivwas carried out by treating the intermediate with either commerciallyavailable acyl chloride or a freshly prepared acyl chloride preparedfrom the reaction of the corresponding carboxylic acid and oxalylchloride as shown in Reaction Scheme 3.

In an alternate method for introducing the R₁ group into the compoundsof structures (1a) and (1b), the intermediate of structure ix wasprepared from isatoic anhydrides and alkylated at the N-1 position in afinal step. The isatoic anhydride was converted into the intermediate ofstructure v by treating it with diethyl malonate. The amination,followed by reaction with hot phosphorous oxychloride, of intermediate vyielded the dichloroquinolinone intermediate of structure vii. Thereaction of intermediate vii with ammonium acetate in acetic acid gaveintermediate of structure viii, which was treated with acyl piperazineto yield the intermediate of structure ix as depicted in Reaction Scheme4.

The alkylation of the N-1 position of intermediate ix yielded thedesired compounds with a different R₁ substitution. The alkylation wascarried out by either heating intermediate ix with potassium carbonateand the corresponding alkyl halide, or by treating the intermediate withsodium hydride and alkyl halide at room temperature, as depicted inReaction Scheme 5.

Acyl and alkylpiperazines suitable for use as intermediates may besynthesized as follows. A solution of freshly distilled thionyl chloride(3.9 ml; 0.053 mol) in methylene dichloride (5 ml) was added dropwise toa stirred solution of 2-thiophenemethanol (4.2 ml; 0.044 mol) andtriethylamine (7.4 ml; 0.05 mol) in methylene dichloride (25 ml) at atemperature kept below 20° C. The temperature was then raised to 40° C.over 1 h, and the solution poured onto crushed ice. The CH₂Cl₂ phase wasseparated and dried over MgSO₄, then added dropwise to a stirredsolution of N—Boc-piperazine (2 g; 0.011 mol) and triethylamine (1.5 ml;0.011 mol) in CH₂Cl₂ (45 ml). See, e.g., Meanwell et al., J. Med. Chem.(1993) Vol. 36., pp. 3251–3264; Carceller et al., J. Med. Chem. (1993)No. 36, pp. 2984–2997. The mixture was stirred overnight at roomtemperature. The solvent was then removed under reduced pressure, andthe residue was extracted with ether. The ether solution was evaporatedunder reduced pressure, and the residue was dissolved in trifluoroaceticacid (TFA) (3.3 ml; 0.043 mol) and kept during 30 min. TFA was removedunder reduced pressure, the residue was triturated with ether, theprecipitate was filtered off and dried in air to yield1-(2-thienylmethyl)piperazine ditrifluoroacetate (3.16 g; 72%). See,e.g., Archer et al., J. Chem. Soc. Perkin Trans. II. (1983) pp. 813–819.

A solution of freshly distilled thionyl chloride (3.9 ml; 0.053 mol) inmethylene dichloride (5 ml) was added dropwise to a stirred solution offurfuryl alcohol (3.8 ml; 0.044 mol) and triethylamine (7.4 ml; 0.05mol) in methylene dichloride (25 ml); the temperature was maintainedbelow 20° C. The mixture was stirred for 1 h, then the solvent wasevaporated, and the residue was dissolved in CH₂Cl₂ (150 ml). Thesolution obtained was added dropwise to a stirred solution ofN—Boc-piperazine (2 g; 0.011 mol) and triethylamine (4 ml; 0.029 mol) inCH₂Cl₂ (45 ml). The mixture was stirred overnight at room temperature,the solvent was removed under reduced pressure, and the residue wasextracted with ether. The ether solution was evaporated under reducedpressure, the residue was dissolved in TFA (3.3 ml; 0.043 mol) andmaintained for 30 min. TFA was removed under reduced pressure, theresidue was triturated with ether, and the black precipitate obtainedwas filtered off. Then, the precipitate was dissolved in 200 ml of MeOH,activated charcoal was added, and the mixture was heated under refluxfor 30 min. Charcoal was filtered off, the solvent was evaporated, theresidue was triturated with ether. The white precipitate obtained wasfiltered off and dried on the air to yield 1-(2-furylmethyl)piperazineditrifluoroacetate (1.64 g; 40%). See, e.g., Lukes et al., CollectionCzechoslov. Chem. Commun. (1954) Vol. 19, pp. 609–610.

Preparation of 4-(Thiophene-2-carbonyl)-piperazine-1-carboxylic acidtert-butyl ester (Compound 595-03)

2-Thiophenecarbonylchloride (2.04 g, 1.49 mL) was added to a solution oftert-butyl-1-piperazinecarboxylate (2.5 g, 13.4 mmol) and DMAP (20 mg)in pyridine (15 mL) at 0° C. under N₂ atmosphere and stirred at roomtemperature for overnight. The mixture was poured into ice water, theprecipitate was filtered, washed several times with water, and dried toyield white solids (3.5 g, 88%). M.P. 76° C. ¹H NMR (DMSO-d₆): δ 1.42(s, 12H), 3.40 (m, 4H), 3.61 (m, 4H), 7.12 (m, 1H), 7.43 (d, J=4.1 Hz,1H), 7.77 (d, J=4.8 Hz, 1H). EIMS m/z 297 (M+1), 319 (M+23). Anal.(C₁₄H₂₀N₂O₃S) C, H, N.

Preparation of Piperazine-1-yl-thiophen-2-yl-methanone (Compound 595-04)

To a solution of 595-03 (3.5 g, 11.8 mmol) in dichloromethane (50 mL)was added trifluoroacetic acid (10 mL). The solution was stirred at roomtemperature for 3 h. The solvent was evaporated under vacuum and theresidue was dissolved in chloroform. The organic phase was washed bysaturated solution of sodium bicarbonate, dried over Na₂SO₄ andevaporated to get 2.20 g (94%) of brown viscous oil. ¹H NMR (DMSO-d₆): δ2.78 (m, 4H), 3.59 (m, 4H), 7.12 (t, J=4.1, 1H), 7.38 (d, J=4.1 Hz, 1H),7.74 (d, J=4.8 Hz, 1H). EIMS m/z 197 (M+1).

N-substituted piperazines of structure vi are useful intermediates inthe preparation of MIF inhibitors. They may be prepared by deprotectionof protected intermediate v (in this case, protected withN-tert-butyloxycarbonyl or “Boc” for purpose of illustration). Theprotected intermediate may be made from the N-protected piperazine iv byaddition of the desired R₄ group. In the above reaction scheme, Z is—CH₂— or —C(═O)—; n is 0, 1 or 2, with the proviso that when n is 0, Zis —C(═O)—; R₄ is selected from the group consisting of —CH₂R₇,—C(═O)NR₅R₆, —C(═O)OR₇, —C(═O)R₇,R₈, and

R₅ and R₆ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl,substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; or R₅ and R₆ takentogether with a nitrogen atom to which they are attached form aheterocycle or substituted heterocycle; R₇ is selected from the groupconsisting of alkyl, substituted alkyl, aryl, substituted aryl,arylalkyl, substituted arylalkyl, heterocycle, substituted heterocycle,heterocyclealkyl, and substituted heterocyclealkyl; and R₈ is selectedfrom the group consisting of hydrogen, alkyl, substituted alkyl, aryl,substituted aryl, arylalkyl, substituted arylalkyl, heterocycle,substituted heterocycle, heterocyclealkyl, and substitutedheterocyclealkyl.

Example 3

The following describes the synthesis of a library of compounds ofgeneral structure 1′(a) and 1′(b) as depicted below. Compounds including“M” in the designation incorporate the CN moiety. Numerical designationsin Table 1 are as given below.

In compounds that have designations including “+i”, R₁₃ is a substituenton the oxygen atom of the quinolone group rather than the nitrogen atom,i.e., a compound of structure 1′(b), as depicted below. The designation“i” appears elsewhere in the preferred embodiments, and refers to asubstituent on the oxygen atom of the quinolone group rather than thenitrogen atom.

Numerical Designations for R₁₁ Functional Groups

Hydrogen Methyl Chlorine 1 2 3

Numerical Designations for R₁₂ Functional Groups

Numerical Designations for R₁₃ Functional Groups

The numerical designations of the MIF inhibitors prepared are providedin Table 1.

TABLE 1 R₁₃ = 1Methyl

1M11 1M12 1M13 1M14 1M15 + i 1M21 1M22 1M23 1M24 1M25 + i 1M31 1M32 1M331M34 1M35 + i 1M41 1M42 1M43 1M44 1M45 + i 1M51 1M52 1M53 1M54 1M55 1M611M62 1M63 1M64 1M65 2M11 2M12 2M13 2M14 2M15 2M21 2M22 2M23 2M24 2M25 +i 2M31 2M32 2M33 2M34 2M35 + i 2M41 2M42 2M43 2M44 2M45 + i 2M51 2M522M53 2M54 2M55 2M61 2M62 2M63 2M64 2M65 + i 3M11 3M12 3M13 3M14 3M15 + i3M21 3M22 3M23 3M24 3M25 3M31 3M32 3M33 3M34 3M35 + i 3M41 3M42 3M433M44 3M45 + i 3M51 3M52 3M53 3M54 3M55 + i 3M61 3M62 3M63 3M64 3M65 + i

Details of reaction schemes for preparing intermediates or MIFinhibitors are provided below.

SYNTHESIS OF REPRESENTATIVE COMPOUNDS Preparation of4-Hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid ethylester (Compound 1)

A solution of diethyl malonate (8.16 g, 51 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 2.24 g, 56 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then heated to90° C. for 30 min. and cooled to room temperature. A solution ofN-methylisatoic anhydride (10 g, 56 mmol) in dimethylacetamide was addedslowly and the mixture was heated overnight at 120° C. The mixture wasthen cooled to room temperature, poured into ice water, and acidified bycold 10% HCl. The solids formed were filtered and washed several timesby water to yield 8.47 g (67%) of white solids. M.P. 67° C. ¹H NMR(DMSO-d₆): δ 1.30 (t, J=7.0 Hz, 3H), 3.53 (s, 3H), 4.32 (q, J=7.0 Hz,2H), 7.30 (t, J=7.5 Hz, 1H), 7.52 (d, J=8.5 Hz, 1H), 7.75 (t, J=8.4 Hz,1H), 8.04 (d, J=7.8 Hz, 1H), 13.03 (s, 1H). EIMS m/z 248 (M+1), 270(M+23). Anal. (C₁₃H₁₃NO₄) C, H, N.

Preparation of4-Hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acidcyclohexylamide (Compound 2)

Cyclohexylamine (2.0 mL, 18.20 mmol) was added to a solution of Compound1 (2.25 g, 9.1 mmol) in toluene (20 mL) and refluxed for 4 h. Thesolution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, and filteredto yield 1.9 g (70%) of white solids. M.P. 145° C. ¹H NMR (DMSO-d₆): δ1.26 (m, 1H), 1.38 (m, 4H), 1.54 (m, 1H), 1.68 (m, 2H), 1.86 (M, 2H),3.62 (s, 3H), 3.87 (m, 1H), 7.37 (t, J=7.5 Hz, 1H), 7.63 (d, J=8.5 Hz,1H), 7.80 (t, J=8.4 Hz, 1H), 8.09 (d, J=7.8 Hz, 1H), 10.46 (s, 1H),17.46 (s, 1H), EIMS m/z 301 (M+1), 323 (M+23). Anal. (C₁₇H₂₀N₂O₃) C, H,N.

Preparation of4-Chloro-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile (Compound3)

A solution of Compound 2 (1.5 g, 5 mmol) in 20 mL phosphorus oxychloridewas heated at 90° C. for 2 h. The solvent was evaporated under reducedpressure. The residue was suspended in ice water and neutralized bysolid sodium bicarbonate. The solids formed were filtered, washed bywater, and purified by flash chromatography eluting with 1% methanol indichloromethane to yield 903 mg (82%) of white solids. M.P. 235° C. ¹HNMR (DMSO-d₆): 3.66 (s, 3H), 7.50 (t, J=7.7 Hz, 1H), 7.74 (d, J=8.6 Hz,1H), 7.91 (t, J=8.7 Hz, 1H), 8.08 (d, J=7.6 Hz, 1H). EIMS m/z 219 (M+1),241 (M+23). Anal. (C₁₇H₇ClN₂O) C, H, N.

Preparation of1-Methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 4)

Piperazine-1-yl-thiophen-2-yl-methanone (600 mg, 3.06 mmol) was added toa solution of Compound 3 (319 mg, 1.46 mmol) in toluene (40 mL) andheated overnight at 120° C. The solvent was removed under vacuum. Theresidue was suspended in water, sonicated, and filtered to yield 540 mg(98%) of white solids. M.P. 247° C. ¹H NMR (DMSO-d₆): δ 3.58 (s, 3H),3.63 (m, 4H), 3.92 (m, 4H), 7.16 (t, J=4.8 Hz, 1H), 7.33 (t, J=7.5 Hz,1H), 7.48 (d, J=3.5 Hz, 1H), 7.59 (d, J=8.5 Hz, 1H), 7.75 (t, J=7.25,2H), 7.79 (d, J=4.8 Hz, 1H) 7.92 (d, J=7.5 Hz, 1H). EIMS m/z 379 (M+1),401 (M+23). Anal. (C₂₀H₁₈N₄O₂S) C, H, N.

Preparation of 1-(4-Fluorobenzyl)-1H-benzo[d][1,3]oxazine-2,4-dione(Compound 5)

A solution of isatoic anhydride (20 g, 122 mmol) in dimethylformamide(DMF) was added to a suspension of NaH (60% in mineral oil, 5.39 g, 135mmol) in DMF and stirred at room temperature for 1 h. Then,4-fluorobenzyl bromide (16.8 mL, 135 mmol) was added and the mixturestirred at room temperature for 4 h. The solution was poured into waterand the solids formed were filtered, washed several times by water anddried. The solids were suspended in hexane, sonicated briefly, filtered,and washed by hexane to yield 30 g (90%) of white solids. M.P. 167° C.¹H NMR (DMSO-d₆): δ 5.27 (s, 2H), 7.17 (t, J=8.8 Hz, 2H), 7.25 (d, J=8.4Hz, 1H), 7.31 (t, J=7.4 Hz, 1H), 7.47 (m, 2H), 7.74 (t, J=7.0 Hz, 1H),8.02 (d, J=7.8 Hz, 1H). Anal. (C₁₅H₁₀FNO₃) C, H, N.

Preparation of1-(4-Fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 6)

A solution of diethyl malonate (8.0 g, 51 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 2.21 g, 55 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then heated to90° C. for 30 min. and cooled to room temperature. A solution ofCompound 5 (15 g, 53 mmol) in dimethylacetamide was added slowly to themixture, which was heated overnight at 120° C. The mixture was cooled toroom temperature, poured into ice water, and acidified by cold 10% HCl.The solids formed were filtered and washed several times by water toyield 13.37 g (78%) of white solids. M.P. 116–120° C. ¹H NMR (DMSO-d₆):δ 1.31 (t, J=7.0 Hz, 3H), 4.36 (q, J=7.0 Hz, 2H), 5.43 (s, 2H), 7.13 (m,2H), 7.23–7.30 (m, 3H), 7.37 (d, J=8.5 Hz, 1H), 7.63 (t, J=7.0 Hz, 1H),8.08 (d, J=7.6 Hz, 1H), 13.20 (s, 1H). EIMS m/z 342 (M+1), 364 (M+23).Anal. (C₁₉H₁₆FNO₄) C, H, N.

Preparation of1-(4-Fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 7)

Cyclohexylamine (0.67 mL, 5.85 mmol) was added to a solution of Compound6 (1.0 g, 2.92 mmol) in toluene (20 mL) and refluxed for 4 h. Thesolution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystalized by ether to yield 1.0 g(87%) of white solids. M.P. 168° C. ¹H NMR (DMSO-d₆): δ 1.26 (m, 1H),1.36 (m, 4H), 1.55 (m, 1H), 1.68 (m, 2H), 1.89 (m, 2H), 3.88 (m, 1H),5.51 (s, 2H), 7.13 (m, 2H), 7.25 (m, 2H), 7.33 (t, J=7.5 Hz, 1H), 7.45(d, J=8.5 Hz, 1H), 7.70 (t, J=8.4 Hz, 1H), 8.12 (d, J=7.9 Hz, 1H), 10.35(s, 1H), 17.66 (s, 1H), EIMS m/z 395 (M+1), 417 (M+23). Anal.(C₂₃H₂₃FN₂O₃) C, H, N.

Preparation of4-Chloro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 8)

A solution of Compound 7 (0.7 g, 1.77 mmol) in 20 mL neat phosphorusoxychloride was heated at 90° C. for 2 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 420 mg (76%) of whitesolids. M.P. 231° C. ¹H NMR (DMSO-d₆): δ 5.54 (s, 2H), 7.13 (m, 2H),7.34 (m, 2H), 7.46 (t, J=7.5 Hz, 1H), 7.55 (d, J=8.5 Hz, 1H), 7.80 (t,J=8.4 Hz, 1H), 8.12 (d, J=7.9 Hz, 1H). EIMS m/z 335 (M+23). Anal.(C₁₇H₁₀ClFN₂O) C, H, N.

Preparation of1-(4-Fluorobenzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 9)

Piperazine-1-yl-thiophen-2-yl-methanone (234 mg, 1.19 mmol) was added toa solution of Compound 8 (170 mg, 0.54 mmol) in toluene (40 mL) andheated overnight at 120° C. The solvent was removed under vacuum. Theresidue was suspended in water, sonicated, and filtered to yield 247 mg(97%) of white solids. M.P. 258° C. ¹H NMR (DMSO-d₆): δ 3.69 (m, 4H),3.94 (m, 4H), 5.47 (s, 2H), 7.16 (m, 3H), 7.28 (m, 3H), 7.43 (d, J=8.5Hz, 1H), 7.50 (d, J=3.3 Hz, 1H), 7.64 (t, J=7.25, 2H), 7.81 (d, J=4.8Hz, 1H) 7.95 (d, J=7.5 Hz, 1H). EIMS m/z 473 (M+1), 495 (M+23). Anal.(C₂₆H₂₁FN₄O₂S) C, H, N.

Preparation of 2-Amino-5-methyl benzoic acid (Compound 10)

To a solution of 5-methyl-2-nitrobenzoic acid (20 g, 110 mmol) inethanol was added 10% Pd/C (1 g). The mixture was stirred overnight atroom temperature under hydrogen atmosphere. The solution was filteredthrough celite and evaporated under reduced pressure to yield 16 g (96%)of white solids. M.P. 162° C. ¹H NMR (DMSO-d₆): δ 2.13 (s, 3H), 6.65 (d,J=8.6 Hz, 1H), 7.06 (dd, J=8.6, 1.8 Hz, 1H), 7.48 (d, J=1.1, 1H). EIMSm/z 174 (M+1), 152 (M+23).

Preparation of 6-Methyl-1H-benzo[d][1,3]oxazine-2,4-dione (Compound 11)

Trichloromethyl chloroformate (36.27 mL, 300 mmol) was added to astirred solution of Compound 10 (41.3 g, 273 mmol) in dry dioxane atroom temperature and the solution was refluxed for 4 h. The solution wascooled in ice bath and the solids formed were filtered. The solids werewashed by ether and dried under vacuum at room temperature to yield 45.5g (94%) of white solids. M.P. 257° C. ¹H NMR (DMSO-d₆): δ 2.32 (s, 3H),7.06 (d, J=8.6 Hz, 1H), 7.56 (dd, J=8.6, 1.8 Hz, 1H), 7.71 (d, J=1.1,1H), 11.63 (s, 1H). EIMS (neg. mode) m/z 176 (M−1), 152 (M+23). Anal.(C₉H₇NO₃) C, H, N.

Preparation of 1-Benzyl-6-methyl-1H-benzo[d][1,3]oxazine-2,4-dione(Compound 12)

A solution of Compound 11 (25 g, 141 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 6.21 g, 155 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat benzyl bromide(19.53 mL, 155 mmol) was added and the solution further stirred at roomtemperature for 3 h. The solution was poured into ice water, and thesolids formed were filtered, washed several times by water, and dried.The solid was suspended in hexane, sonicated briefly, filtered, andwashed by hexane to yield 36.5 g (97%) of white solids. M.P. 150° C. ¹HNMR (DMSO-d₆): δ 2.32 (s, 3H), 5.27 (s, 2H), 7.15 (d, J=8.7 Hz, 1H),7.26–7.39 (m, 5H), 7.54 (dd, J=1.5, 8.7 Hz, 1H), 7.83 (d, J=1.5 Hz, 1H).Anal. (C₁₆H₁₃NO₃) C, H, N.

Preparation of1-(4-Fluorobenzyl-6-methyl-1H-benzo[d][1,3]oxazine-2,4-dione (Compound13)

A solution of Compound 11 (5 g, 28 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 1.24 g, 31 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat 4-fluorobenzylbromide (3.81 mL, 31 mmol) was added, and the solution further stirredat room temperature for 3 h. The solution was poured into ice water andthe solids formed were filtered, washed several times by water, anddried. The solids were suspended in hexane, sonicated briefly, filtered,and washed by hexane to yield 6.3 g (79%) of white solids. M.P. 153° C.¹H NMR (DMSO-d₆): δ 2.32 (s, 3H), 5.24 (s, 2H), 7.14–7.17 (m, 3H), 7.44(m, 2H), 7.54 (dd, J=1.7, 8.2 Hz, 1H), 7.83 (d, J=1.2 Hz, 1H). Anal.(C₁₆H₁₂FNO₃) C, H, N.

Preparation of 1,6-Dimethyl-1H-benzo[d][1,3]oxazine-2,4-dione (Compound14)

A solution of Compound 11 (5 g, 28 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 1.24 g, 31 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, methyl iodide (1.92mL, 31 mmol) was added and further stirred at room temperature for 3 h.The solution was poured into ice water and the solids formed werefiltered, washed several times by water, and dried. The solids weresuspended in hexane, sonicated briefly, filtered, and washed by hexaneto yield 4.9 g (74%) of white solids. M.P. 153° C. ¹H NMR (DMSO-d₆): δ2.32 (s, 3H), 3.52 (s, 3H), 7.38 (d, J=8.6 Hz, 1H), 7.54 (dd, J=1.7, 8.2Hz, 1H), 7.83 (d, J=1.2 Hz, 1H).

Preparation of1-Benzyl-4-hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 15)

Neat diethyl malonate (19.07 mL, 125 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 5.52 g, 138 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then heated to90° C. for 30 min and cooled to room temperature. A solution of Compound12 (36.8 g, 138 mmol) in dimethylacetamide was added slowly and themixture was heated overnight at 110° C. The mixture was cooled to roomtemperature, poured into ice water, and acidified by cold 10% HCl. Thesolids formed were filtered, washed several times by water, and dried atroom temperature under vacuum to yield 41 g (97%) of white solids. M.P.113° C. ¹H NMR (DMSO-d₆): δ 1.31 (t, J=7.5 Hz, 3H), 2.33 (s, 3H), 4.35(q, J=7.5 Hz, 2H), 5.43 (s, 2H), 7.15–7.30 (m, 6H), 7.43 (dd, J=1.6, 8.7Hz, 1H), 7.85 (d, J=1.5 Hz, 1H). EIMS m/z 338 (M+1), 360 (M+23). Anal.(C₂₀H₁₉NO₄) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-4-hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 16)

Neat diethyl malonate (3.04 mL, 20 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 0.88 g, 22 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min and cooled to room temperature. Asolution of Compound 13 (6.3 g, 22 mmol) in dimethylacetamide was addedslowly and the mixture heated overnight at 110° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered, washed several times by water,and dried at room temperature under vacuum to yield 5.1 g (71%) of whitesolids. M.P. 130° C. ¹H NMR (DMSO-d₆): δ 1.31 (t, J=7.0 Hz, 3H), 2.34(s, 3H), 4.37 (q, J=7.5 Hz, 2H), 5.94 (s, 2H), 7.09–7.32 (m, 5H), 7.45(dd, J=1.6, 8.7 Hz, 1H), 7.86 (d, J=1.5 Hz, 1H). EIMS m/z 356 (M+1), 378(M+23). Anal. (C₂₀H₁₈FNO₄) C, H, N.

Preparation of4-Hydroxy-1,6-dimethyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acidethyl ester (Compound 17)

Neat diethyl malonate (2.28 g, 14.26 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 628 mg, 15.69 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min. and cooled to room temperature. Asolution of Compound 14 (10 g, 56 mmol) in dimethylacetamide was addedslowly and the mixture heated overnight at 120° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered and washed several times bywater to yield 3.26 g (87%) of white solids. M.P. 132° C. ¹H NMR(DMSO-d₆): δ 1.30 (t, J=6.9 Hz, 3H), 2.50 (s, 3H), 3.50 (s, 3H), 4.33(q, J=6.9 Hz, 2H), 7.41 (d, J=8.6 Hz, 1H), 7.56 (dd, J=1.7, 8.5 Hz, 1H),7.82 (d, J=1.7 Hz, 1H), 13.03 (s, 1H). EIMS m/z 262 (M+1), 284 (M+23).

Preparation of1-Benzyl-4-hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 18)

Cyclohexylamine (3.41 mL, 29.64 mmol) was added to a solution ofCompound 15 (5.0 g, 14.82 mmol) in toluene (50 mL) and refluxed for 4 h.The solution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 5.5 g(95%) of white solids. M.P. 87° C. ¹H NMR (DMSO-d₆): δ 1.22 (m, 1H),1.36 (m, 4H), 1.56 (m, 1H), 1.67 (m, 2H), 1.88 (m, 2H), 2.36 (s, 3H),3.87 (m, 1H), 5.51 (s, 2H), 7.14–7.33 (m, 6H), 7.47 (d, J=8.6 Hz, 1H),7.90 (s, 1H), 10.44 (s, 1H). EIMS m/z 391 (M+1).

Preparation of1-(4-Fluorobenzyl)-4-hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 19)

Cyclohexylamine (3.22 mL, 28.14 mmol) was added to a solution ofCompound 16 (5.0 g, 14.07 mmol) in toluene (50 mL) and refluxed for 4 h.The solution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 5.3 g(93%) of yellow solids. M.P. 156° C. ¹H NMR (DMSO-d₆): δ 1.24 (m, 1H),1.37 (m, 4H), 1.57 (m, 1H), 1.68 (m, 2H), 1.88 (m, 2H), 2.36 (s, 3H),3.87 (m, 1H), 5.49 (s, 2H), 7.11 (m, 2H), 7.22 (m, 2H), 7.36 (d, J=8.7Hz, 1H), 7.51 (dd, J=1.6, 8.7 Hz, 1H), 7.90 (d, J=1.6 Hz, 1H), 10.39 (s,1H). EIMS m/z 409 (M+1), 431 (M+23).

Preparation of4-Hydroxy-1,6-dimethyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acidcyclohexylamide (Compound 20)

Cyclohexylamine (2.85 mL, 24.95 mmol) was added to a solution ofCompound 17 (3.26 g, 12.47 mmol) in toluene (50 mL) and refluxed for 4h. The solution was cooled and the solvent was evaporated under vacuum.The residue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 3.8 g(97%) of white solids. M.P. 218° C. ¹H NMR (DMSO-d₆): δ 1.28 (m, 1H),1.36 (m, 4H), 1.55 (m, 1H), 1.68 (m, 2H), 1.87 (m, 2H), 2.40 (s, 3H),3.59 (s, 3H), 3.87 (m, 1H), 7.51 (d, J=8.7 Hz, 1H), 7.61 (d, J=8.7 Hz,1H), 7.86 (s, 1H), 10.49 (s, 1H). EIMS m/z 315 (M+1), 337 (M+23).

Preparation of1-Benzyl-4-chloro-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 21)

A solution of Compound 18 (5 g, 12.80 mmol) in 30 mL neat phosphorusoxychloride was heated at 100° C. for 3 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 1.51 g (38%) of yellowsolids. M.P. 219° C. ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H), 5.54 (s, 2H),7.23–7.26 (m, 3H), 7.29–7.32 (m, 2H), 7.45 (d, J=8.8 Hz, 1H), 7.61 (dd,J=1.5, 8.8 Hz, 1H), 7.89 (d, J=1.5 Hz, 1H). EIMS m/z 309 (M+1), 331(M+23).

Preparation of4-Chloro-1-(4-fluorobenzyl)-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 22)

A solution of Compound 19 (5 g, 12.24 mmol) in 30 mL neat phosphorusoxychloride was heated at 100° C. for 3 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 1.90 g (47%) of yellowsolids. M.P. 206° C. ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H), 5.52 (s, 2H),7.12–7.15 (m, 2H), 7.30–7.33 (m, 2H), 7.46 (d, J=8.7 Hz, 1H), 7.64 (dd,J=1.2, 8.7 Hz, 2H), 7.89 (d, J=1.2 Hz, 1H). EIMS m/z 327 (M+1).

Preparation of4-Chloro-1,6-dimethyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 23)

A solution of Compound 20 (3 g, 9.5 mmol) in 30 mL neat phosphorusoxychloride was heated at 100° C. for 3 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 1.2 g (54%) of yellowsolids. M.P. 241° C. ¹H NMR (DMSO-d₆): δ 2.44 (s, 3H), 3.64 (s, 3H),7.62 (d, J=8.7 Hz, 1H), 7.73 (dd, J=1.5, 8.7 Hz, 1H), 7.85 (d, J=1.5 Hz,1H). EIMS m/z 255 (M+1).

Preparation of1-Benzyl-6-methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 24)

Piperazine-1-yl-thiophen-2-yl-methanone (490 mg, 2.5 mmol) was added toa solution of Compound 20 (309 mg, 1 mmol) in toluene (20 mL) and heatedovernight at 110° C. The solvent was removed under vacuum. The residuewas suspended in water, sonicated, and filtered. The crude product waspurified by flash chromatography eluting with 0–2% methanol indichloromethane gradient to yield 362 mg (77%) of white solids. M.P.183° C. ¹H NMR (DMSO-d₆): δ 2.36 (s, 3H), 3.69 (m, 4H), 3.94 (m, 4H),5.47 (s, 2H), 7.16–7.20 (m, 4H), 7.23–7.33 (m, 3H), 7.44 (d, J=8.8 Hz,1H), 7.50 (d, J=3.8 Hz, 1H), 7.70 (s, 1H), 7.81 (d, J=4.8 Hz, 1H). EIMSm/z 491 (M+23). Anal. (C₂₇H₂₄N₄O₂S) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-6-methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 25)

Piperazine-1-yl-thiophen-2-yl-methanone (490 mg, 2.5 mmol) was added toa solution of Compound 21 (327 mg, 1 mmol) in toluene (20 mL) and heatedovernight at 110° C. The solvent was removed under vacuum. The residuewas suspended in water, sonicated, and filtered. The crude product waspurified by flash chromatography eluting with 0–2% methanol indichloromethane gradient to yield 210 mg (43%) of white solids. M.P.274° C. ¹H NMR (DMSO-d₆): δ 2.37 (s, 3H), 3.68 (m, 4H), 3.94 (m, 4H),5.45 (s, 2H), 7.12–7.18 (m, 3H), 7.24–7.27 (m, 2H), 7.33 (d, J=8.6 Hz,1H), 7.45 (d, J=8.8 Hz, 1H), 7.50 (d, J=3.5 Hz, 1H), 7.70 (s, 1H), 7.81(d, J=4.8 Hz, 1H). EIMS m/z 509 (M+23). Anal. (C₂₇H₂₃FN₄O₂S) C, H, N,

Preparation of1-Benzyl-6-methyl-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 26)

A solution of Compound 21 (1.2 g, 3.9 mmol) in dichloromethane was addedslowly to a stirred solution of piperazine (1.67 g, 19.4 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was removed under reduced pressure. Theresidue was taken in water, sonicated briefly, and filtered. The solidswere dissolved in ethyl acetate and washed by water. The organic layerwas dried over Na₂SO₄ and concentrated to yield 1.34 g (96%) of yellowsolids. M.P. 152° C. ¹H NMR (DMSO-d₆): δ 2.34 (s, 3H), 2.94 (m, 4H),3.54 (m, 4H), 5.44 (s, 2H), 7.16–7.20 (m, 2H), 7.22 (d, J=7.6 Hz, 1H),7.28–7.31 (m, 3H), 7.40 (d, J=8.8 Hz, 1H), 7.64 (s, 1H). EIMS m/z 359(M+1).

Preparation of1-(4-Fluoro-benzyl)-6-methyl-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 27)

A solution of Compound 22 (1.6 g, 4.9 mmol) in dichloromethane was addedslowly to a stirred solution of piperazine (2.2 g, 24.5 mmol) in CH₂Cl₂at room temperature and further stirred overnight. The solvent wasremoved under reduced pressure. The residue was taken in water,sonicated briefly, and filtered. The solids were dissolved in ethylacetate and washed by water. The organic layer was dried over Na₂SO₄ andconcentrated to yield 1.30 g (72%) of yellow solids. M.P. 121° C. ¹H NMR(DMSO-d₆): δ 2.35 (s, 3H), 2.96 (m, 4H), 3.54 (m, 4H), 5.42 (s, 2H),7.11–7.17 (m, 2H), 7.23–7.26 (m, 2H), 7.44 (dd, J=1.2, 8.6 Hz, 1H), 7.64(s, 1H). EIMS m/z 377 (M+1).

Preparation of1,6-Dimethyl-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 28)

A solution of Compound 23 (1 g, 4.3 mmol) in dichloromethane was addedslowly to a stirred solution of piperazine (1.1 g, 12.9 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was removed under reduced pressure. Theresidue was taken in water, sonicated briefly, and filtered. The solidswere dissolved in ethyl acetate and washed by water. The organic layerwas dried over Na₂SO₄ and concentrated to yield 1.07 g (88%) yellowsolids. M.P. 274° C. ¹H NMR (DMSO-d₆): δ 2.39 (s, 3H), 2.94 (m, 4H),3.48 (m, 4H), 3.54 (s, 3H), 7.45 (d, J=8.6 Hz, 1H), 7.56 (dd, J=1.3, 8.6Hz, 1H), 7.64 (s, 1H). EIMS m/z 283 (M+1).

Preparation of1-Benzyl-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-6-methyl-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 29)

2-Furoyl chloride (118 μL, 1.2 mmol) was added to a stirred solution ofCompound 26 (287 mg, 0.8 mmol) in pyridine (5 mL) under argon at 0° C.The solution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried andrecrystallized by hexane and ether to yield 340 mg (90%) of whitesolids. M.P. 146° C. ¹H NMR (DMSO-d₆): δ 2.36 (s, 3H), 3.69 (m, 4H),3.97 (m, 4H), 5.47 (s, 2H), 6.66 (t, J=2.5 Hz, 1H), 7.09 (d, J=3.4 Hz,1H), 7.18 (d, J=7.3 Hz, 2H), 7.23 (d, J=7.4 Hz, 1H), 7.29–7.33 (m, 3H),7.44 (d, J=1.3, 8.7 Hz, 1H), 7.71 (s, 1H), 7.89 (s, 1H). EIMS m/z 475(M+23). Anal. (C₂₇H₂₄N₄O₃) C, H, N.

Preparation of1-(4-Fluorob-enzyl)-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-6-methyl-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 30)

2-Furoyl chloride (118 μL, 1.2 mmol) was added to a stirred solution ofCompound 27 (300 mg, 0.8 mmol) in pyridine (5 mL) under argon at 0° C.The solution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by hexane and ether to yield 320 mg (85%) of whitesolids. M.P. 276° C. ¹H NMR (DMSO-d₆): δ 2.37 (s, 3H), 3.69 (m, 4H),3.97 (m, 4H), 5.45 (s, 2H), 6.67 (dd, J=1.7, 3.7 Hz, 1H), 7.09 (d, J=3.4Hz, 1H), 7.12 (m, 2H), 7.26 (m, 2H), 7.35 (d, J=8.7 Hz, 1H), 7.45 (d,J=8.8 Hz, 1H), 7.71 (s, 1H), 7.89 (s, 1H). EIMS m/z 493 (M+23). Anal.(C₂₇H₂₃FN₄O₃) C, H, N.

Preparation of4-[4-(Furan-2-carbonyl)-piperazin-1-yl]-1,6-dimethyl-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 31)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 28 (282 mg, 1.0 mmol) in pyridine (5 mL) under argon at 0° C.The solution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethyl acetate to yield 221 mg (59%) of white solids.M.P. 231° C. ¹H NMR (DMSO-d₆): δ 2.41 (s, 3H), 3.56 (s, 3H), 3.63 (m,4H), 3.95 (m, 4H), 6.66 (dd, J=1.5, 3.6 Hz, 1H), 7.08 (d, J=3.4 Hz, 1H),7.48 (d, J=8.7 Hz, 1H), 7.59 (dd, J=1.2, 8.8 Hz, 1H), 7.69 (s, 1H), 7.88(s, 1H). EIMS m/z 399 (M+23). Anal. (C₂₁H₂₀N₄O₃) C, H, N.

Preparation of1,6-Dimethyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 32)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 28 (282 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 277 mg (70%) ofwhite solids. M.P. 214° C. ¹H NMR (DMSO-d₆): δ 2.41 (s, 3H), 3.55 (s,3H), 3.63 (m, 4H), 3.92 (m, 4H), 7.16 (t, J=4.1 Hz, 1H), 7.47 (m, 2H),7.56 (d, J=8.7 Hz, 1H), 7.68 (s, 1H), 7.80 (d, J=4.9 Hz, 1H). EIMS m/z393 (M+1), 415 (M+23). Anal. (C₂₁H₂₀N₄O₂S) C, H, N.

Preparation of 5-Fluoro-2-nitrobenzoic acid (Compound 33)

3-Fluorobenzoic acid (1 g, 7.13 mmol) was dissolved in concentratedH₂SO₄ (2 ml) by warming slightly above room temperature. The solutionwas cooled to 0° C. Fuming nitric acid (539 mg, 8.56 mmol) was addedslowly to the solution while keeping the temperature below 0° C. Thesolution was stirred at 0° C. for 3 h. The solution was poured into icewater, the solid formed were filtered, washed by cold water, and driedto yield 1.2 g (92%) of white solids. M.P. 122° C. ¹H NMR (DMSO-d₆):7.60 (dt, J=2.9, 8.5 Hz, 1H), 7.71 (dd, J=2.9, 8.6 Hz, 1H), 8.13 (dd,J=4.8, 8.8 Hz, 1H). EIMS m/z 186 (M+1).

Preparation of 2-Amino-5-fluoro benzoic acid (Compound 34)

A solution of Compound 33 (10 g, 54 mmol) in ethanol (100 mL) wasstirred under hydrogen in the presence of 10% Pd/C (0.5 g) at roomtemperature for 4 h. The solution was filtered through celite. Thesolvent was evaporated under reduced pressure to yield 8.2 g (98%) ofwhite solids. M.P. 142° C. ¹H NMR (DMSO-d₆): 6.71 (dd, J=4.9, 8.9 Hz,1H), 7.15 (dt, J=2.9, 8.4 Hz, 1H), 7.37 (dd, J=2.9, 9.8 Hz, 1H), 8.60(s, 1H). EIMS m/z 156 (M+1).

Preparation of 6-Fluoro-1H-benzo[d][1,3]oxazine-2,4-dione (Compound 35)

Trichloromethyl chloroformate (7.01 mL, 58.13 mmol) was added to astirred solution of Compound 34 (8.2 g, 52.85 mmol) in dry dioxane atroom temperature and the solution was refluxed for 4 h. The solution wascooled in an ice bath and the solids formed were filtered. The solidswere washed by ether and dried under vacuum at room temperature to yield9.1 g (96%) of white solids. M.P. 240° C. ¹H NMR (DMSO-d₆): δ 7.19 (dd,J=4.2, 8.9 Hz, 1H), 7.63–7.71 (m, 1H), 11.77 (s, 1H). EIMS (neg. mode)m/z 180 (M−1). Anal. (C₈H₄FNO₃) C, H, N.

Preparation of 1-Benzyl-6-fluoro-1H-benzo[d][1,3]oxazine-2,4-dione(Compound 36)

A solution of Compound 35 (3 g, 16.57 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 729 mg, 18.23 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat benzyl bromide(2,17 mL, 18.23 mmol) was added and the solution was further stirred atroom temperature for 3 h. The solution was poured into ice water and thesolids formed were filtered, washed several times by water, and dried.The solids were suspended in hexane, sonicated briefly, filtered, andwashed by hexane to yield 1.88 g (42%) of white solids. M.P. 95° C. ¹HNMR (DMSO-d₆): δ 5.29 (s, 2H), 7.26 (m, 2H), 7.35 (m, 5H), 7.40 (m, 2H),7.64 (dt, J=2.9, 8.4 Hz, 1H), 7.82 (dd, J=3.2, 8.0 Hz, 1H). Anal.(C₁₆H₁₃NO₃) C, H, N.

Preparation of 6-Fluoro-1-(4-fluorobenzyl)-1H-benzo[d][1,3]oxazine-2,4-dione (Compound 37)

A solution of Compound 35 (3 g, 16.57 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 729 mg, 18.22 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat 4-fluorobenzylbromide (2.28 mL, 18.23 mmol) was added and the solution further stirredat room temperature for 3 h. The solution was poured into ice water andthe solids formed were filtered, washed several times by water, anddried. The solids were suspended in hexane, sonicated briefly, filtered,and washed by hexane to yield 3.23 g (67%) of white solids. M.P. 107° C.¹H NMR (DMSO-d₆): δ 5.27 (s, 2H), 7.19 (m, 2H), 7.29 (dd, J=3.9, 9.0 Hz,1H), 7.47 (m, 2H), 7.65 (td, J=5.5, 9.0 Hz, 1H), 7,81 (dd, J=2.9, 7.9Hz, 1H). Anal. (C₁₅H₉F₂NO₃) C, H, N.

Preparation of 6-Fluoro-1-methyl-1H-benzo[d][1,3]oxazine-2,4-dione(Compound 38)

A solution of Compound 35 (3 g, 16.57 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 0.729 g, 18.23 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat methyl iodide(1.14 mL, 18.23 mmol) was added and the solution further stirred at roomtemperature for 3 h. The solution was poured into ice water and thesolids formed were filtered, washed several times by water, and dried.The solids were suspended in hexane, sonicated briefly, filtered, andwashed by hexane to yield 1.84 g (57%) of white solids. M.P. 133° C. ¹HNMR (DMSO-d₆): δ 3.90 (s, 3H), 7.51 (dd, J=4.0, 8.8 Hz, 1H), 7.77 (m,12). Anal. (C₉H₆FNO₃) C, H, N.

Preparation of1-Benzyl-6-fluoro-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 39)

Neat diethyl malonate (0.89 mL, 5.8 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 256 mg, 6.41 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min and cooled to room temperature. Asolution of Compound 36 (1.74 g, 6.41 mmol) in dimethylacetamide wasadded slowly to the mixture, which was heated overnight at 110° C. Themixture was cooled to room temperature, poured into ice water, andacidified by cold 10% HCl. The solids formed were filtered, washedseveral times by water, and dried at room temperature under vacuum toyield 1.4 g (64%) of white solids. M.P. 129° C. ¹H NMR (DMSO-d₆): δ 1.30(t, J=6.9 Hz, 3H), 4.34 (q, J=6.9 Hz, 2H), 5.46 (s, 2H), 7.17–7.24 (m,5H), 7.38 (dd, J=4.6, 9.6 Hz, 1H), 7.50 (td, J=2.9, 8.3 Hz, 1H), 7.80(dd, J=3.1, 9.4 Hz, 1H). EIMS m/z 342 (M+1), 364 (M+23). Anal.(C₁₉H₁₆FNO₄) C, H, N.

Preparation of6-Fluoro-1-(Fluoro-benzyl)-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 40)

Neat diethyl malonate (1.2 mL, 8.0 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 352 mg, 8.8 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min and cooled to room temperature. Asolution of Compound 37 (2.5 g, 8.8 mmol) in dimethylacetamide was addedslowly and heated overnight at 110° C. The mixture was cooled to roomtemperature, poured into ice water, and acidified by cold 10% HCl. Thesolids formed were filtered, washed several times by water, and dried atroom temperature under vacuum to yield 2.5 g (87%) of white solids. M.P.123° C. ¹H NMR (DMSO-d₆): δ 1.31 (t, J=7.0 Hz, 3H), 4.37 (q, J=7.5 Hz,2H), 5.43 (s, 2H), 7.19 (m, 2H), 7.29 (dd, J=3.9, 9.0 Hz, 1H), 7.47 (m,2H), 7.65 (td, J=5.5, 9.0 Hz, 1H), 7.81 (dd, J=2.9, 7.9 Hz, 1H). EIMSm/z 360 (M+1), 382 (M+23). Anal. (C₁₉H₁₅F₂NO₄) C, H, N.

Preparation of6-Fluoro-4-hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 41)

Neat diethyl malonate (1.27 g, 8.4 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 372 mg, 9.3 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min. and cooled to room temperature. Asolution of Compound 38 (1.8 g, 9.3 mmol) in dimethylacetamide was addedslowly and the mixture heated overnight at 120° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered and washed several times bywater to yield 1.3 g (53%) of white solids. M.P. 131° C. ¹H NMR(DMSO-d₆): δ 1.30 (t, J=6.9 Hz, 3H), 3.54 (s, 3H), 4.31 (q, J=6.9 Hz,2H), 7.65 (dd, J=4.6, 9.3 Hz, 1H), 7.64 (dd, J=2.9, 9.1 Hz, 1H), 7.76(dd, J=2.9, 9.3 Hz, 1H), 12.80 (s, 1H). EIMS m/z 266 (M+1), 288 (M+23).

Preparation of1-Benzyl-6-fluoro-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 42)

Cyclohexylamine (2.95 mL, 25.78 mmol) was added to a solution ofCompound 39 (4.4 g, 12.89 mmol) in toluene (50 mL) and refluxed for 3 h.The solution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 4.0 g(78%) of white solids. M.P. 130–134° C. ¹H NMR (DMSO-d₆): δ 1.37 (m,5H), 1.56 (m, 1H), 1.67 (m, 2H), 1.88 (m, 2H), 3.87 (m, 1H), 5.29 (s,2H), 7.17 (m, 2H), 7.23 (m, 1H), 7.33 (m, 1H), 7.46 (dd, J=3.9, 9.1 Hz,1H), 7.57 (td, J=2.9, 8.3 Hz, 1H), 7.81 (dd, J=2.9, 8.8 Hz, 1H). EIMSm/z 394 (M+1).

Preparation of6-Fluoro-1-(4-fluorobenzyl)-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 43)

Cyclohexylamine (3.25 mL, 28.38 mmol) was added to a solution ofCompound 40 (5.1 g, 14.19 mmol) in toluene (50 mL) and refluxed for 4 h.The solution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 5.0 g(86%) of white solids. M.P. 118° C. ¹H NMR (DMSO-d₆): δ 1.18 (m, 2H),1.35 (m, 3H), 1.57 (m, 1H), 1.67 (m, 2H), 1.88 (m, 2H), 3.86 (m, 1H),5.48 (s, 2H), 7.13 (m, 2H), 7.24 (m, 2H), 7.35 (dd, J=3.9, 9.0 Hz, 1H),7.53 (td, J=5.5, 9.0 Hz, 1H), 7.80 (dd, J=2.9, 7.9 Hz, 1H). EIMS m/z 413(M+1).

Preparation of6-Fluoro-4-hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 44)

Cyclohexylamine (1.98 mL, 17.34 mmol) was added to a solution ofCompound 41 (2.3 g, 8.67 mmol) in toluene (50 mL) and refluxed for 4 h.The solution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 2.38 g(97%) of white solids. M.P. 193° C. ¹H NMR (DMSO-d₆): δ 1.28 (m, 1H),1.36 (m, 4H), 1.55 (m, 1H), 1.68 (m, 2H), 1.87 (m, 2H), 3.62 (s, 3H),3.90 (m, 1H), 7.70 (m, 3H), 7.77 (d, J=8.0 Hz, 1H), 10.41 (s, 1H). EIMSm/z 341 (M+23).

Preparation of1-Benzyl-4-chloro-6-fluoro-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 45)

A solution of Compound 42 (3.5 g, 8.86 mmol) in 30 mL neat phosphorusoxychloride was heated at 90° C. for 5 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 1.29 g (47%) of yellowsolids. M.P. 228° C. ¹H NMR (DMSO-d₆): δ 5.29 (s, 2H), 7.27 (m, 4H),7.32 (m, 2H), 7.58 (dd, J=4.3, 9.3 Hz, 1H), 7.71 (td, J=2.4, 8.0 Hz,1H), 7.91 (dd, J=2.9, 8.9 Hz, 1H). EIMS m/z 335 (M+23).

Preparation of4-Chloro-6-fluoro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 46)

A solution of Compound 43 (5.1 g, 12.36 mmol) in 20 ml neat phosphorusoxychloride was heated at 100° C. for 5 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 2.05 g (50%) of yellowsolids. M.P. 236° C. ¹H NMR (DMSO-d₆): δ 5.54 (s, 2H), 7.14 (m, 2H),7.33 (m, 2H), 7.59 (dd, J=4.0, 9.1 Hz, 1H), 7.72 (td, J=2.8, 7.9 Hz,1H), 7.91 (dd, J=2.8, 8.9 Hz, 1H). EIMS m/z 331 (M+1).

Preparation of4-Chloro-6-fluoro-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 47)

A solution of Compound 44 (2 g, 6.28 mmol) in 20 ml neat phosphorusoxychloride was heated at 90° C. for 5 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 0.80 g (56%) of yellowsolids. M.P. 258° C. ¹H NMR (DMSO-d₆): δ 3.67 (s, 3H), 7.78–7.89 (m,3H). EIMS m/z 259 (M+23).

Preparation of1-Benzyl-6-fluoro-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 48)

A solution of Compound 45 (1.0 g, 3.2 mmol) in dichloromethane was addedslowly to a stirred solution of piperazin (826 mg g, 9.59 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was removed under reduced pressure. Theresidue was taken in water sonicated briefly and filtered. The solid wasdissolved in ethyl acetate and washed by water. The organic layer wasdried over Na₂SO₄ and concentrated to yield 1.1 g (96%) yellow solids.M.P. 162° C. ¹H NMR (DMSO-d₆): δ 2.70 (s, 1H), 2.94 (m, 4H), 3.53 (m,4H), 5.47 (s, 2H), 7.19–7.25 (m, 3H), 7.42 (m, 2H), 7.51 (dd, J=4.3, 9.3Hz, 1H), 7.54 (td, J=2.4, 8.0 Hz, 1H), 7.58 (dd, J=2.9, 8.9 Hz, 1H).EIMS m/z 363 (M+1).

Preparation of6-Fluoro-1-(4-Fluoro-benzyl)-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 49)

A solution of Compound 46 (2.0 g, 6.04 mmol) in dichloromethane wasadded slowly to a stirred solution of piperazine (1.56 g, 18.21 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was removed under reduced pressure. Theresidue was taken in water, sonicated briefly, and filtered. The solidswere dissolved in ethyl acetate and washed by water. The organic layerwas dried over Na₂SO₄ and concentrated to yield 2.18 g (95%) yellowsolids. M.P. 240° C. ¹H NMR (DMSO-d₆): δ 2.84 (s, 1H), 2.94 (m, 4H),3.54 (m, 4H), 5.45 (s, 2H), 7.15 (m, 2H), 7.27 (m, 2H), 7.44 (dd, J=4.4,9.3 Hz, 1H), 7.53 (td, J=2.4, 8.0 Hz, 1H), 7.58 (dd, J=2.9, 8.9 Hz, 1H).EIMS m/z 381 (M+1).

Preparation of6-Fluoro-1-methyl-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 50)

A solution of Compound 47 (750 mg, 3.17 mmol) in dichloromethane wasadded slowly to a stirred solution of piperazine (819 mg, 9.50 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was removed under reduced pressure. Theresidue was taken in water, sonicated briefly, and filtered. The solidswere dissolved in ethyl acetate and washed by water. The organic layerwas dried over Na₂SO₄ and concentrated to yield 780 mg (86%) of yellowsolids. M.P. 211° C. ¹H NMR (DMSO-d₆): δ 2.91 (m, 4H), 3.47 (m, 4H),3.56 (s, 3H), 7.54 (m, 1H), 7.63 (m, 2H). EIMS m/z 287 (M+1).

Preparation of1-Benzyl-6-fluoro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 51)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 48 (363 mg, 1 mmol) in pyridine (5 mL) under argon at 0° C. Thesolution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethyl acetate to yield 332 mg (73%) of white solids.M.P. 209° C. ¹H NMR (DMSO-d₆): δ 3.69 (m, 4H), 3.97 (m, 4H), 5.49 (s,2H), 6.66 (m, 1H), 7.09 (d, J=3.3 Hz, 1H), 7.22 (m, 3H), 7.32 (m, 2H),7.45 (dd, J=4.7, 9.5 Hz, 1H), 7.55 (m, 1H), 7.69 (dd, J=2.8, 9.7 Hz,1H), 7.88 (s, 1H). EIMS m/z 479 (M+23). Anal. (C₂₆H₂₁FN₄O₃) C, H, N.

Preparation of6-Fluoro-1-(4-fluoro-benzyl)-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 52)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 49 (380 mg, 1 mmol) in pyridine (5 mL) under argon at 0° C. Thesolution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethylacetate to yield 282 mg (85%) of white solids.M.P. 248° C. ¹H NMR (DMSO-d₆): δ 3.68 (m, 4H), 3.96 (m, 4H), 5.47 (s,2H), 6.66 (dd, J=1.6, 3.6 Hz, 1H), 7.08 (d, J=3.4 Hz, 1H), 7.15 (m, 2H),7.28 (m, 2H), 7.47 (dd, J=4.7, 9.6 Hz, 1H), 7.55 (m, 1H), 7.69 (dd,J=2.8, 9.7 Hz, 1H), 7.88 (s, 1H). EIMS m/z 497 (M+23). Anal.(C₂₆H₂₀F₂N₄O₃) C, H, N.

Preparation of6-Fluoro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-1-methyl-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 53)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 50 (286 mg, 1.0 mmol) in pyridine (5 mL) under argon at 0° C.The solution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethyl acetate to yield 333 mg (87%) of white solids.M.P. 263° C. ¹H NMR (DMSO-d₆): δ 3.58 (s, 3H), 3.62 (m, 4H), 3.95 (m,4H), 6.66 (d, J=3.6 Hz, 1H), 7.08 (d, J=3.4 Hz, 1H), 7.65 (m, 3H), 7.88(s, 1H). EIMS m/z 403 (M+23). Anal. (C₂₀H₁₇FN₄O₃) C, H, N.

Preparation of1-Benzyl-6-fluoro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 54)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 48 (362 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 359 mg (76%) ofwhite solids. M.P. 246° C. ¹H NMR (DMSO-d₆): δ 3.67 (m, 4H), 3.94 (m,4H), 5.49 (s, 2H), 7.16 (m, 1H), 7.20–7.26 (m, 3H), 7.45 (dd, J=4.6, 9.4Hz, 1H), 7.50 (d, J=3.9 Hz, 1H), 7.55 (td, J=2.7, 9.2 Hz, 1H), 7.66 (dd,J=2.8, 9.7 Hz, 1H), 7.80 (d, J=5.1 Hz, 1H). EIMS m/z 473 (M+1). Anal.(C₂₆H₂₁FN₄O₂S) C, H, N.

Preparation of6-Fluoro-1-(fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 55)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 49 (380 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 294 mg (60%) ofwhite solids. M.P. 211° C. ¹H NMR (DMSO-d₆): δ 3.67 (m, 4H), 3.94 (m,4H), 5.47 (s, 2H), 7.15 (m, 3H), 7.27 (m, 2H), 7.46 (m, 2H), 7.54 (td,J=2.6, 9.2 Hz, 1H), 7.67 (dd, J=2.8, 9.7 Hz, 1H), 7.80 (d, J=5.1 Hz,1H). EIMS m/z 491 (M+1). Anal. (C₂₆H₂₀F₂N₄O₂S) C, H, N.

Preparation of6-Fluoro-1-methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 56)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 50 (286 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 390 mg (98%) ofwhite solids. M.P. 286° C. ¹H NMR (DMSO-d₆): δ 3.58 (s, 3H), 3.62 (m,4H), 3.92 (m, 4H), 7.16 (dd, J=3.5, 5.0, 1H), 7.50 (d, J=3.2 Hz, 1H),7.63–7.68 (m, 3H), 7.80 (d, J=4.8 Hz, 1H). EIMS m/z 397 (M+1). Anal.(C₂₀H₁₇FN₄O₂S) C, H, N.

Preparation of 2-Amino-5-chloro benzoic acid (Compound 57)

To a solution of 5-chloro-2-nitrobenzoic acid (20 g, 110 mmol) inethanol was added freshly activated raney nickel (2 g). The mixture wasstirred overnight at room temperature under hydrogen atmosphere. Thesolution was filtered through celite and evaporated under reducedpressure to yield 16 g (96%) of white solids. ¹H NMR (DMSO-d₆): δ 6.77(d, J=8.9 Hz, 1H), 7.24 (dd, J=2.9, 8.9 Hz, 1H), 7.62 (d, J=2.9 Hz, 1H),8.7 (b, 3H); EIMS: 170 (M−H).

Preparation of 6-Chloro-1H-benzo[d][1,3]oxazine-2,4-dione (Compound 58)

Trichloromethyl chloroformate (4.8 mL, 300 mmol) was added to a stirredsolution of Compound 57 (6.84 g, 40 mmol) in dry dioxane at roomtemperature and the solution was refluxed for 4 h. The solution wascooled in an ice bath and the solids formed were filtered. The solidswere washed by ether and dried under vacuum at room temperature to yield7.3 g (92%) of white solids. ¹H NMR (DMSO-d₆): δ 7.47 (d, J=8.6 Hz, 1H),7.70 (dd, J=8.6, 1.8 Hz, 1H), 7.82 (d, J=1.1, 1H), 11.63 (s, 1H).

Preparation of 1-Benzyl-6-chloro-1H-benzo[d][1,3]oxazine-2,4-dione(Compound 59)

A solution of Compound 58 (4.9 g, 25 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 1.2 g, 30 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat benzyl bromide(3.78 mL, 30 mmol) was added and the solution further stirred at roomtemperature for 3 h. The solution was poured into ice water and thesolids formed were filtered, washed several times by water, and dried.The solids were suspended in hexane, sonicated briefly, filtered, andwashed by hexane to yield 6.5 g (90%) of white solids. ¹H NMR (DMSO-d₆):δ 5.45 (s, 2H), 7.17 (d, J=8.7 Hz, 1H), 7.26–7.39 (m, 5H), 7.65 (dd,J=1.5, 8.7 Hz, 1H), 8.02 (d, J=1.5 Hz, 1H).

Preparation of 6-Chloro-1-(4-Fluorobenzyl)-1H-benzo[d][1,3]oxazine-2,4-dione (Compound 60)

A solution of Compound 58 (5 g, 25 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 1.24 g, 31 mmol) in DMF andfurther stirred at room temperature for 1 h. Then, neat 4-fluorobenzylbromide (3.81 mL, 31 mmol) was added and the solution further stirred atroom temperature for 3 h. The solution was poured into ice water and thesolids formed were filtered, washed several times by water, and dried.The solids were suspended in hexane, sonicated briefly, filtered, andwashed by hexane to yield 7.3 g (96%) of white solids. ¹H NMR (DMSO-d₆):δ 5.45 (s, 2H), 7.14–7.17 (m, 3H), 7.44 (m, 2H), 7.64 (dd, J=1.7, 8.2Hz, 1H), 7.80 (d, J=1.2 Hz, 1H).

Preparation of 1,6-Dimethyl-1H-benzo[d][1,3]oxazine-2,4-dione (Compound61)

A solution of Compound 58 (5 g, 25 mmol) in DMF was added slowly to asuspension of NaH (60% in mineral oil, 1.24 g, 31 mmol) in DMF, and thesolution was further stirred at room temperature for 1 h. Then, methyliodide (1.92 mL, 31 mmol) was added and the solution was further stirredat room temperature for 3 h. The solution was poured into ice water andthe solids formed were filtered, washed several times by water, anddried. The solids were suspended in hexane, sonicated briefly, filtered,and washed by hexane to yield 4.6 g (75%) of white solids. ¹H NMR(DMSO-d₆): δ 3.52 (s, 3H), 7.54 (d, J=8.2 Hz, 1H), 7.74 (dd, J=1.7, 8.2Hz, 1H), 7.80 (d, J=1.2 Hz, 1H).

Preparation of1-Benzyl-6-chloro-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 62)

Neat diethyl malonate (19.07 mL, 125 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 5.52 g, 138 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min then cooled to room temperature. Asolution of Compound 59 (39.88 g, 138 mmol) in dimethylacetamide wasadded slowly and the mixture heated overnight at 110° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered, washed several times by water,and dried at room temperature under vacuum to yield 38 g (86%) of whitesolids. ¹H NMR (DMSO-d₆): δ 1.31 (t, J=7.2 Hz, 3H), 4.32 (q, J=7.2 Hz,2H), 5.45 (s, 2H), 7.17 (d, J=7.2 Hz, 2H), 7.2 (m, 2H), 7.31 (t, J=6.8Hz, 1H), 7.37 (d, J=9.2 Hz, 1H), 7.65 (dd, J=2.8, 9.2 Hz, 1H), 8.02 (d,J=2.4 Hz, 1H), 12.90 (b, 1H); EIMS: 358 (M+H).

Preparation of6-Chloro-1-(4-Fluoro-benzyl)-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 63)

Neat diethyl malonate (3.04 mL, 20 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 0.88 g, 22 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min and cooled to room temperature. Asolution of Compound 60 (6.7 g, 22 mmol) in dimethylacetamide was addedslowly and the mixture heated overnight at 110° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered, washed several times by water,and dried at room temperature under vacuum to yield 6.4 g (85%) of whitesolids. ¹H NMR (DMSO-d₆): δ 1.31 (t, J=6.8 Hz, 3H), 4.34 (q, J=6.8 Hz,2H), 5.43 (s, 2H), 7.1 (m, 2H), 7.2 (m, 2H), 7.40 (d, J=9.2, 1H), 7.66(dd, J=2.4, 9.2 Hz, 1H), 8.02 (d, J=2.8 Hz, 1H), 13.00 (b, 1H); EIMS:376 (M+H).

Preparation of6-Chloro-4-Hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid ethyl ester (Compound 64)

Neat diethyl malonate (2.28 g, 14.26 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 628 mg, 15.69 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min. then cooled to room temperature. Asolution of Compound 61 (12 g, 56 mmol) in dimethylacetamide was addedslowly and the mixture heated overnight at 120° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered and washed several times bywater to yield 3.96 g (97%) of white solids. ¹H NMR (DMSO-d₆): δ 1.29(t, J=6.9 Hz, 3H), 3.52 (s, 3H), 4.30 (q, J=6.9 Hz, 2H), 7.54 (d, J=9.1Hz, 1H), 7.74 (dd, J=1.6, 8.9 Hz, 1H), 8.0 (m, 1H), 12.80 (b, 1H); EIMS:282 (M+H).

Preparation of1-Benzyl-6-chloro-4-hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 65)

Cyclohexylamine (1.27 mL, 11.17 mmol) was added to a solution ofCompound 62 (2.0 g, 5.6 mmol) in toluene (50 mL) and refluxed for 4 h.The solution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 2.23 g(96%) of white solids. M.P. 158° C. ¹H NMR (DMSO-d₆): δ 1.22 (m, 2H),1.35 (m, 3H), 1.56 (m, 1H), 1.67 (m, 2H), 1.88 (m, 2H), 3.87 (m, 1H),5.51 (s, 2H), 7.16 (m, 2H), 7.24 (m, 1H), 7.32 (m, 2H), 7.42 (d, J=9.0Hz, 1H), 7.70 (d, J=9.0 Hz, 1H), 7.80 (d, J=2.1 Hz, 1H), 10.36 (s, 1H).EIMS m/z 411 (M+1).

Preparation of6-Chloro-1-(4-fluorobenzyl)-4-hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 66)

Cyclohexylamine (1.83 mL, 16 mmol) was added to a solution of Compound63 (3.0 g, 8 mmol) in toluene (50 mL) and refluxed for 4 h. The solutionwas cooled and the solvent was evaporated under vacuum. The residueobtained was suspended in water, briefly sonicated, and filtered. Thecrude product was recrystallized by ether to yield 3.1 g (90%) of whitesolids. M.P. 157° C. ¹H NMR (DMSO-d₆): δ 1.21 (m, 2H), 1.35 (m, 3H),1.55 (m, 1H), 1.67 (m, 2H), 1.88 (m, 2H), 2.36 (s, 3H), 3.87 (m, 1H),5.49 (s, 2H), 7.13 (m, 2H), 7.23 (m, 2H), 7.44 (d, J=9.3 Hz, 1H), 7.70(dd, J=2.6, 9.4 Hz, 1H), 7.80 (d, J=2.8 Hz, 1H), 10.33 (s, 1H).

Preparation of4-Chloro-6-hydroxy-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylicacid cyclohexylamide (Compound 67)

Cyclohexylamine (1.62 mL, 14.2 mmol) was added to a solution of Compound64 (2.0 g, 7.1 mmol) in toluene (50 mL) and refluxed for 4 h. Thesolution was cooled and the solvent was evaporated under vacuum. Theresidue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 2.27 g(67%) of white solids. M.P. 186° C. ¹H NMR (DMSO-d₆): δ 1.28 (m, 1H),1.38 (m, 4H), 1.54 (m, 1H), 1.69 (m, 2H), 1.87 (m, 2H), 3.60 (s, 3H),3.87 (m, 1H), 7.66 (d, J=9.0 Hz, 1H), 7.83 (d, J=9.0 Hz, 1H), 7.98 (s,1H), 10.39 (s, 1H). EIMS m/z 335 (M+1).

Preparation of1-Benzyl-4,6-dichloro-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 68)

A solution of Compound 65 (2 g, 4.86 mmol) in 20 mL neat phosphorusoxychloride was heated to 90° C. for 5 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 810 mg (51%) of yellowsolids. M.P. 243° C. ¹H NMR (DMSO-d₆): δ 5.55 (s, 2H), 7.25 (m, 3H),7.32 (m, 2H), 7.56 (d, J=9.0 Hz, 1H), 7.85 (dd, J=2.1, 9.0 Hz, 1H), 7.89(d, J=2.1 Hz, 1H). EIMS m/z 352 (M+23).

Preparation of4,6-Dichloro-1-(4-fluorobenzyl)-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 69)

A solution of Compound 66 (3 g, 7 mmol) in 30 mL neat phosphorusoxychloride was heated at 90° C. for 5 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 1.2 g (50%) of yellowsolids. M.P. 252° C. ¹H NMR (DMSO-d₆): δ 5.53 (s, 2H), 7.15 (m, 2H),7.33 (m, 2H), 7.56 (d, J=9.0 Hz, 1H), 7.86 (dd, J=2.1, 9.0 Hz, 1H), 8.08(d, J=2.1 Hz, 1H).

Preparation of4,6-Dichloro-1-methyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 70)

A solution of Compound 67 (2.2 g, 6.57 mmol) in 20 mL neat phosphorusoxychloride was heated at 90° C. for 5 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 0.8 g (48%) of yellowsolids. M.P. 256° C. ¹H NMR (DMSO-d₆): δ 3.65 (s, 3H), 7.60 (d, J=9.0Hz, 1H), 7.94 (dd, J=2.1, 9.0 Hz, 1H), 8.06 (d, J=2.1 Hz, 1H). EIMS m/z254 (M+1).

Preparation of1-Benzyl-6-chloro-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 71)

A solution of Compound 68 (0.8 g, 2.43 mmol) in dichloromethane wasadded slowly to a stirred solution of piperazine (628 mg g, 7.29 mmol)in dichloromethane at room temperature. The solution was stirredovernight at room temperature. The solvent was then removed underreduced pressure. The residue was taken in water, sonicated briefly, andfiltered. The solids were dissolved in ethyl acetate and washed bywater. The organic layer was dried over Na₂SO₄ and concentrated to yield0.9 g (98%) of yellow solids. M.P. 156° C. ¹H NMR (DMSO-d₆): δ 2.94 (m,4H), 3.55 (m, 4H), 5.47 (s, 2H), 7.19 (m, 2H), 7.24 (m, 1H), 7.31 (m,2H), 7.39 (d, J=8.9 Hz, 1H), 7.64 (dd, J=2.6, 8.9 Hz, 1H), 7.81 (d,J=2.6 Hz, 1H). EIMS m/z 379 (M+1).

Preparation of6-Chloro-1-(4-fluoro-benzyl)-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 72)

A solution of Compound 69 (1.2 g, 3.48 mmol) in dichloromethane wasadded slowly to a stirred solution of piperazine (0.9 g, 10.45 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was then removed under reducedpressure. The residue was taken in water, sonicated briefly, andfiltered. The solids were dissolved in ethyl acetate and washed bywater. The organic layer was dried over Na₂SO₄ and concentrated to yield1.36 (98%) of yellow solids. M.P. 189° C. ¹H NMR (DMSO-d₆): δ 2.94 (m,4H), 3.54 (m, 4H), 5.43 (s, 2H), 7.14 (m, 2H), 7.26 (m, 2H), 7.43 (d,J=9.0 Hz, 1H), 7.67 (dd, J=2.5, 9.0 Hz, 1H), 7.81 (d, J=2.5 Hz, 1H).EIMS m/z 397 (M+1).

Preparation of6-Chloro-1-methyl-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 73)

A solution of Compound 70 (0.8 g, 3.16 mmol) in dichloromethane wasadded slowly to a stirred solution of piperazine (819 mg, 9.50 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was then removed under reducedpressure. The residue was taken in water, sonicated briefly, andfiltered. The solids were dissolved in ethyl acetate and washed bywater. The organic layer was dried over Na₂SO₄ and concentrated to yield950 mg (99%) of yellow solids. M.P. 223° C. ¹H NMR (DMSO-d₆): δ 2.92 (m,4H), 3.48 (m, 4H), 3.55 (s, 3H), 7.60 (d, J=8.8 Hz, 1H), 7.77 (m, 2H).EIMS m/z 303 (M+1).

Preparation of1-Benzyl-6-chloro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 74)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 71 (379 mg, 1 mmol) in pyridine (5 mL) under argon at 0° C. Thesolution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethyl acetate to yield 361 mg (76%) of white solids.M.P. 221° C. ¹H NMR (DMSO-d₆): δ 3.70 (m, 4H), 3.97 (m, 4H), 5.48 (s,2H), 6.67 (dd, J=2.0, 3.6 Hz, 1H), 7.10 (d, J=3.6 Hz, 1H), 7.20 (m, 2H),7.25 (m, 1H), 7.27 (m, 2H), 7.42 (d, J=9.2 Hz, 1H), 7.70 (dd, J=2.4, 9.2Hz), 7.90 (s, 1H). EIMS m/z 496 (M+23). Anal. (C₂₆H₂₁ClN₄O₃) C, H, N.

Preparation of6-Chloro-1-(4-fluoro-benzyl)-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 75)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 72 (397 mg, 1 mmol) in pyridine (5 mL) under argon at 0° C. Thesolution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethylacetate to yield 212 mg (43%) of white solids.M.P. 253° C. ¹H NMR (DMSO-d₆): δ 3.69 (m, 4H), 3.96 (m, 4H), 5.45 (s,2H), 6.66 (m, 1H), 7.08 (d, J=3.6 Hz, 1H), 7.20 (m, 2H), 7.27 (m, 2H),7.46 (d, J=9.0 Hz, 1H), 7.69 (dd, J=2.4, 9.0 Hz), 7.89 (s, 1H). EIMS m/z514 (M+23). Anal. (C₂₆H₂₀ClFN₄O₃) C, H, N.

Preparation of6-Chloro-4-[4-(furan-2-carbonyl)-piperazin-1-yl]-1-methyl-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 76)

2-Furoyl chloride (148 μL, 1.5 mmol) was added to a stirred solution ofCompound 73 (303 mg, 1.0 mmol) in pyridine (5 mL) under argon at 0° C.The solution was allowed to come to room temperature and further stirredovernight. The solution was poured into ice water and the solids formedwere filtered. The solids were washed by excess water, dried, andrecrystallized by ethyl acetate to get 271 mg (68%) of white solids.M.P. 228° C. ¹H NMR (DMSO-d₆): δ 3.56 (s, 3H), 3.64 (m, 4H), 3.94 (m,4H), 6.65 (m, 1H), 7.08 (d, J=3.4 Hz, 1H), 7.60 (d, J=9.0 Hz, 1H), 7.79(dd, J=1.9, 9.0 Hz, 1H), 7.86 (m, 2H). EIMS m/z 419 (M+23). Anal.(C₂₀H₁₇ClN₄O₃) C, H, N.

Preparation of1-Benzyl-6-chloro-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 77)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 71 (379 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 398 mg (82%) ofwhite solids. M.P. 266° C. ¹H NMR (DMSO-d₆): δ 3.70 (m, 4H), 3.93 (m,4H), 5.48 (s, 2H), 7.15 (m, 1H), 7.19–7.26 (m, 3H), 7.32 (m, 2H), 7.42(d, J=8.9 Hz, 1H), 7.51 (d, J=4.0 Hz, 1H), 7.68 (dd, J=2.6, 8.9 Hz, 1H),7.80 (d, J=4.0 Hz, 1H), 7.88 (s, 1H). EIMS m/z 512 (M+23). Anal.(C₂₆H₂₁ClN₄O₂S) C, H, N.

Preparation of6-Chloro-1-(4-fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 78)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 72 (397 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 280 mg (55%) ofwhite solids. M.P. 253° C. ¹H NMR (DMSO-d₆): δ 3.70 (m, 4H), 3.93 (m,4H), 5.46 (s, 2H), 7.13–7.17 (m, 3H), 7.27 (m, 2H), 7.44 (d, J=9.0 Hz,1H), 7.51 (d, J=4.0 Hz, 1H), 7.68 (dd, J=2.6, 9.0 Hz, 1H), 7.79 (d,J=4.0 Hz, 1H), 7.89 (s, 1H). EIMS m/z 508 (M+1). Anal. (C₂₆H₂₀FClN₄O₂S)C, H, N.

Preparation of6-Chloro-1-methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 79)

2-Thiophene carbonyl chloride (160 μL, 1.5 mmol) was added to a stirredsolution of Compound 73 (303 mg, 1.0 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and recrystallized by ethyl acetate to yield 341 mg (83%) ofwhite solids. M.P. 262° C. ¹H NMR (DMSO-d₆): δ 3.57 (s, 3H), 3.64 (m,4H), 3.91 (m, 4H), 7.16 (m, 1H), 7.50 (d, J=3.2 Hz, 1H), 7.62 (d, J=9.0Hz, 1H), 7.79 (m, 2H), 7.86 (s, 1H). EIMS m/z 414 (M+1). Anal.(C₂₀H₁₇ClN₄O₂S) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-(piperazin-1-yl)-1,2-dihydro-quinolin-3-carbonitrile(Compound 80)

A solution of Compound 8 (15 g, 48 mmol) in dichloromethane was addedslowly to a stirred solution of piperazine (12 g, 144 mmol) indichloromethane at room temperature. The solution was stirred overnightat room temperature. The solvent was removed under reduced pressure. Theresidue was taken in water, sonicated briefly, and filtered. The solidwas dissolved in ethyl acetate and washed by water. The organic layerwas dried over Na₂SO₄ and concentrated to yield 17.17 g (98%) of yellowsolids. ¹H NMR (DMSO-d₆): δ 2.8 (m, 2H), 3.0 (m, 2H), 3.6 (m, 2H), 3.7(m, 2H), 5.45 (s, 2H), 7.1 (m, 2H), 7.3 (m, 3H), 7.4 (m, 1H), 7.6 (m,1H), 7.90 (t, J=8.2 Hz, 1H); EIMS: 363 (M+H).

Acylation at Piperazine Moiety

The compounds referred to as Compound 81 through 102 were prepared byapplying either General Procedure A or General Procedure B as describedbelow. General Procedure A was employed to prepare the title compoundsfrom the commercially available corresponding acid chlorides, whereasGeneral Procedure B was employed to prepare the title compounds fromcommercially available acids.

General Procedure A

The corresponding acid chloride (1.25 mmol) was added to a stirredsolution of Compound 80 (300 mg, 0.82 mmol) in pyridine (5 mL) underargon at 0° C. The solution was allowed to come to room temperature andfurther stirred overnight. The solution was poured into ice water andthe solids formed were filtered. The solids were washed by excess water,dried, and purified by flash chromatography eluting with 0–2% MeOH in aCH₂Cl₂ gradient.

General Procedure B

Oxalyl chloride (1.66 mmol) and DMF (2 drops) were added sequentially toa stirred solution of the corresponding acid (1.25 mmol) in CH₂Cl₂ atroom temperature, then further stirred for 2 h under argon atmosphere.The solvent was removed under vacuum at room temperature to yield thedry corresponding acid chloride. A solution of Compound 80 (300 mg, 0.82mmol) in dry pyridine was added to the residue under argon atmosphereand briefly sonicated. The solution was stirred overnight at roomtemperature under argon atmosphere. The solution was poured into icewater and the solids formed were filtered. The solids were washed byexcess water, dried, and purified by flash chromatography eluting with0–2% MeOH in a CH₂Cl₂ gradient.

Preparation of4-(4-Benzoyl-piperazin-1-yl)-1-(4-fluoro-benzyl)-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 81)

The title compound was prepared by applying General Procedure A to yield269 mg (72%) of white solids. M.P. 242° C. ¹H NMR (DMSO-d₆): δ 3.66 (m,4H), 3.92 (m, 4H), 5.46 (s, 2H), 7.14 (m, 3H), 7.28 (m, 3H), 7.42 (d,J=9.0 Hz, 1H), 7.48 (m, 5H), 7.63 (m, 1H), 7.94 (dd, J=1.5, 9.0 Hz, 1H).EIMS m/z 467 (M+1). Anal. (C₂₈H₂₃FN₄O₂) C, H, N.

Preparation of4-(4-Cyclopentanecarbonyl-piperazin-1-yl)-1-(4-fluoro-benzyl)-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 82)

The title compound was prepared by General Procedure A to yield 216 mg(58%) of white solids. M.P. 233° C. ¹H NMR (DMSO-d₆): δ 1.56–1.75 (m,8H), 3.07 (m, 1H), 3.56 (m, 4H), 3.80 (m, 4H), 5.46 (s, 2H), 7.14 (m,2H), 7.28 (m, 3H), 7.42 (d, J=9.0 Hz, 1H), 7.64 (m, 1H), 7.94 (dd,J=1.5, 9.0 Hz, 1H). EIMS m/z 459 (M+1). Anal. (C₂₇H₂₇FN₄O₂) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(2-thiophen-2-yl-acetyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 83)

The title compound was prepared by applying General Procedure A to yield72 mg (18%) of white solids. M.P. 199° C. ¹H NMR (DMSO-d₆): δ 3.58 (m,4H), 3.82 (m, 4H), 4.05 (s, 2H), 5.46 (s, 2H), 6.97 (m, 2H), 7.14 (m,2H), 7.28 (m, 3H), 7.40 (m, 2H), 7.48 (m, 5H), 7.63 (m, 1H), 7.92 (dd,J=1.5, 9.0 Hz, 1H). EIMS m/z 487.4 (M+1). Anal. (C₂₇H₂₃FN₄O₂S) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-4-[4-(isoxazole-5-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 84)

The title compound was prepared by applying General Procedure A to yield213 mg (58%) of white solids. M.P. 253° C. ¹H NMR (DMSO-d₆): δ 3.70 (m,4H), 3.84 (m, 4H), 5.47 (s, 2H), 7.03 (d, J=2.0 Hz, 1H), 7.13 (m, 2H),7.27 (m, 3H), 7.45 (d, J=8.4 Hz, 1H). 7.64 (m, 1H), 7.93 (dd, J=1.5, 8.0Hz, 1H), 8.79 (d, J=2.0 Hz, 1H). EIMS m/z 458 (M+1). Anal. (C₂₅H₂₀FN₅O₃)C, H, N.

Preparation of4-[4-(4-Fluorobenzoyl)-piperazin-1-yl]-1-(4-fluoro-benzyl)-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 85)

The title compound was prepared by applying General Procedure A to yield341 mg (88%) of white solids. M.P. 272° C. ¹H NMR (DMSO-d₆): δ 3.66 (m,4H), 3.84 (m, 4H), 5.46 (s, 2H), 7.13 (m, 2H), 7.31 (m, 5H), 7.42 (d,J=8.0 Hz, 1H), 7.55 (m, 2H), 7.65 (m, 1H), 7.93 (dd, J=1.2, 8.4 Hz, 1H).EIMS m/z 485 (M+1). Anal. (C₂₈H₂₂F₂N₄O₂) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(pyridine-4-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 86)

The title compound was prepared by applying General Procedure A to yield273 mg (73%) of white solids. M.P. 274° C. ¹H NMR (DMSO-d₆): δ 3.60 (m,4H), 3.86 (m, 4H), 5.46 (s, 2H), 7.12 (m, 2H), 7.28 (m, 3H), 7.42 (d,J=8.0 Hz, 1H), 7.47 (m, 2H), 7.61 (m, 1H), 7.90 (d, J=7.2 Hz, 1H), 8.71(dd, J=1.2, 4.4 Hz, 1H). EIMS m/z 468 (M+1). Anal. (C₂₇H₂₂FN₅O₂) C, H,N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(piperidine-1-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 87)

The title compound was prepared by applying General Procedure B to yield243 mg (62%) of white solids. M.P. 223° C. ¹H NMR (DMSO-d₆): δ 1.51 (m,6H), 3.19 (m, 4H), 3.40 (m, 4H), 3.62 (m, 4H), 5.46 (s, 2H), 7.12 (m,2H), 7.28 (m, 3H), 7.41 (d, J=8.8 Hz, 1H), 7.64 (m, 2H), 7.92 (d, J=8.0Hz, 1H). EIMS m/z 474 (M+1). Anal. (C₂₇H₂₈FN₅O₂) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(Pyrrolidine-1-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 88)

The title compound was prepared by applying General Procedure B to yield252 mg (67%) of white solids. M.P. 232° C. ¹H NMR (DMSO-d₆): δ 1.78 (m,4H), 3.44 (m, 4H), 3.61 (m, 4H), 5.46 (s, 2H), 7.12 (m, 2H), 7.27 (m,3H), 7.42 (d, J=8.8 Hz, 1H), 7.62 (m, 2H), 7.93 (d, J=8.0 Hz, 1H). EIMSm/z 459 (M+1). Anal. (C₂₆H₂₆FN₅O₂) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-sulfonyl)-piperazine-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 89)

The title compound was prepared by applying General Procedure B to yield332 mg (79%) of yellow solids. M.P. 226° C. ¹H NMR (DMSO-d₆): δ 3.25 (m,4H), 3.73 (m, 4H), 5.44 (s, 2H), 7.13 (m, 2H), 7.26 (m, 3H), 7.41 (d,J=8.4 Hz, 1H), 7.61 (m, 2H), 7.71 (m, 1H), 7.80 (d, J=8.0 Hz, 1H), 8.12(d, J=4.5 Hz, 1H). EIMS m/z 509 (M+1). Anal. (C₂₅H₂₁FN₄O₃S₂) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-4-[4-(furan-3-carbonyl)-piperazine-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 90)

The title compound was prepared by applying General Procedure B to yield183 mg (48%) of white solids. M.P. 261° C. ¹H NMR (DMSO-d₆): δ 3.65 (m,4H), 3.86 (m, 4H), 5.47 (s, 2H), 6.73 (s, 1H), 7.13 (m, 2H), 7.28 (m,3H), 7.43 (d, J=8.4 Hz, 1H), 7.64 (m, 1H), 7.78 (s, 1H), 7.92 (dd,J=1.2, 8.4 Hz, 1H), 8.12 (s, 1H). EIMS m/z 457 (M+1). Anal.(C₂₆H₂₁FN₄O₃) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-4-[4-(1-methyl-1-H-pyrrole-2-carbonyl)-piperazine-1-yl]-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 91)

The title compound was prepared by applying General Procedure B to yield233 mg (60%) of white solids. M.P. 236° C. ¹H NMR (DMSO-d₆): δ 3.66 (m,4H), 3.92 (m, 4H), 5.47 (s, 2H), 6.06 (dd, J=2.4, 3.6 Hz, 1H), 6.40 (d,J=4.0 Hz, 1H), 6.93 (s, 1H), 7.15 (m, 2H), 7.29 (m, 3H), 7.43 (d, J=8.4Hz, 1H), 7.62 (m, 1H), 7.94 (dd, J=1.2, 8.0 Hz, 1H). EIMS m/z 470 (M+1).Anal. (C₂₇H₂₄FN₅O₂) C, H, N.

Preparation of4-[4-(5-Acetyl-thiophene-2-carbonyl)-piperazine-1-yl]-1-(4-fluoro-benzyl)-2-oxo-1,2-dihydro-quinolin-3-carbonitrile(Compound 92)

The title compound was prepared by applying General Procedure B to yield259 mg (60%) of white solids. M.P. 269° C. ¹H NMR (DMSO-d₆): δ 3.70 (m,4H), 3.90 (m, 4H), 5.47 (s, 2H), 7.12 (m, 2H), 7.29 (m, 3H), 7.44 (d,J=8.4 Hz, 1H), 7.53 (d, J=3.6 Hz, 1H), 7.64 (m, 1H), 7.94 (m, 2H). EIMSm/z 515 (M+1). Anal. (C₂₈H₂₃FN₄O₃S) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(thiophene-3-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 93)

The title compound was prepared by applying General Procedure B to yield312 mg (79%) of white solids. M.P. 251° C. ¹H NMR (DMSO-d₆): δ 3.66 (m,4H), 3.82 (m, 4H), 5.46 (s, 2H), 7.12 (m, 2H), 7.28 (m, 3H), 7.48 (d,J=8.4 Hz, 1H), 7.61 (m, 2H), 7.87 (m, 1H), 7.92 (d, J=8.4 Hz, 1H). EIMSm/z 473 (M+1). Anal. (C₂₆H₂₁FN₄O₂S) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(pyridine-2-carbonyl)-piperazine-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 94)

The title compound was prepared by applying General Procedure B to yield123 mg (32%) of white solids. M.P. 231° C. ¹H NMR (DMSO-d₆): δ 3.63 (m,2H), 3.72 (m, 4H), 3.96 (m, 2H), 5.47 (s, 2H), 6.73 (s, 1H), 7.14 (m,2H), 7.28 (m, 3H), 7.42 (d, J=8.4 Hz, 1H), 7.50 (m, 1H), 7.68 (m, 2H),7.96 (m, 2H), 8.64 (dd, J=0.8, 4.8 Hz, 1H). EIMS m/z 468 (M+1). Anal.(C₂₇H₂₂FN₅O₂) C, H, N.

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(pyrazine-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 95)

The title compound was prepared by applying General Procedure B. MP:145–156° C.; ¹H-NMR (DMSO-d₆): 3.8 (m, 8H), 5.47 (s, 2H), 7.15 (t, J=8.8Hz, 2H), 7.3 (m, 3H), 7.44 (d, J=8.4 Hz, 1H), 7.64 (t, J=7.8 Hz, 1H),7.95 (dd, J=1.2, 8.4 Hz, 1H), 8.7 (m, 1H), 8.78 (d, J=2.4 Hz, 1H), 8.94(d, J=1.6 Hz, 1H); EIMS: 469 (M+H).

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(quinoline-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 96)

The title compound was prepared by applying General Procedure B. MP:144–157° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 6H), 4.0 (m, 2H), 5.47 (s, 2H),7.3 (m, 3H), 7.43 (d, J=8.4 Hz, 1H), 7.63 (t, J=7.2 Hz, 1H), 7.71 (t,J=7.6 Hz, 1H), 7.78 (d, J=8.4 Hz, 1H), 7.86 (t, J=7.6 Hz, 1H), 7.97 (dd,J=1.2, 8.4 Hz, 1H), 8.07 (d, J=9.2 Hz, 2H), 8.55 (d, J=8.4 Hz, 1H);EIMS: 518 (M+H).

Preparation of1-(4-Fluoro-benzyl)-4-[4-(5-methyl-isoxazole-3-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 97)

The title compound was prepared by applying General Procedure B. MP:130–137° C.; ¹H-NMR (DMSO-d₆): 2.48 (s, 3H), 3.7 (m, 4H), 3.9 (m, 4H),5.47 (s, 2H), 6.53 (s, 1H), 7.15 (t, J=9.2 Hz, 2H), 7.3 (m, 3H), 7.44(d, J=8.4 Hz, 1H), 7.64 (t, J=7.8 Hz, 1H), 7.94 (dd, J=1.2, 8.4 Hz, 1H);EIMS: 472 (M+H).

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(tetrahydro-furan-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 98)

The title compound was prepared by applying General Procedure B. MP:123–135° C.; ¹H-NMR (DMSO-d₆): 1.8 (m, 2H), 2.1 (m, 2H), 3.6 (m, 4H),3.8 (m, 6H), 4.7 (m, 1H), 5.47 (s, 2H), 7.15 (d, J=8.8 Hz, 2H), 7.3 (m,3H), 7.43 (d, J=8.4 Hz, 1H). 7.64 (t, J=7.8 Hz, 1H), 7.93 (dd, J=1.2,8.4 Hz, 1H); EIMS: 461 (M+H).

Preparation of4-[4-(Benzo[1,3]dioxole-5-carbonyl)-piperazin-1-yl]-1-(4-fluoro-benzyl)-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 99)

The title compound was prepared by applying General Procedure B. MP:140–160° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 8H), 5.47 (s, 2H), 6.09 (s, 2H),7.1 (m, 1H), 7.15 (t, J=7.8 Hz, 2H), 7.3 (m, 3H), 7.43 (d, J=8.4 Hz,1H), 7.63 (t, J=7.8 Hz, 1H), 7.92 (dd, J=1.2, 8.0 Hz, 1H); EIMS: 511(M+H).

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(4-trifluoromethyl-benzoyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 100)

The title compound was prepared by applying General Procedure B. MP:181–185° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 8H), 5.47 (s, 2H), 7.1 (m, 2H),7.43 (d, J=8.4 Hz, 1H), 7.6 (m, 1H), 7.7 (m, 1H), 7.8 (m, 1H), 7.9 (m,2H), 7.91 (dd, J=1.2, 8.4 Hz, 1H); EIMS: 535 (M+H).

Preparation of1-(4-Fluoro-benzyl)-4-[4-(1H-imidazole-4-carbonyl)-piperazin-1-yl]-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 101)

The title compound was prepared by applying General Procedure B. MP:176–183° C.; ¹H-NMR (DMSO-d₆): 3.67 (s, 4H), 4.3 (m, 4H), 5.47 (s, 2H),7.3 (m, 3H), 7.45 (d, J=8.4 Hz, 1H), 7.6 (m, 2H), 7.76 (d, J=1.2 Hz,1H), 7.97 (dd, J=1.2, 8.0 Hz, 1H); EIMS: 457 (M+H).

Preparation of1-(4-Fluoro-benzyl)-2-oxo-4-[4-(tetrahydro-thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 102)

The title compound was prepared by applying General Procedure B. MP:133–140° C.; ¹H-NMR (DMSO-d₆): 1.9 (m, 1H), 2.0 (m, 1H), 2.1 (m, 1H),2.3 (m, 1H), 2.9 (m, 2H), 3.7 (m, 8H), 4.32 (t, J=5.6 Hz, 1H), 5.47 (s,2H), 7.15 (t, J=8.0 Hz, 2H), 7.3 m), 3H), 7.43 (d, J=8.4 Hz, 1H), 7.64(t, J=7.6 Hz, 1H), 7.93 (d, J=8.0 Hz, 1H); EIMS: 477 (M+H).

Preparation of4-Hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acid ethylester (Compound 103)

Neat.diethyl malonate (18.05 g, 112.7 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 4.96 mg, 124 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min. then cooled to room temperature. Asolution of Compound 11 (22 g, 124 mmol) in dimethylacetamide was addedslowly and the solution heated overnight at 110° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered and washed several times bywater to yield 10.2 g (36%) of white solids. M.P. 242° C. ¹HNMR(DMSO-d₆): δ 1.30 (t, J=7.2 Hz, 3H), 2.35 (s, 3H), 4.34 (q, J=7.2 Hz,2H), 7.19 (d, J=8.4 Hz, 1H), 7.46 (dd, J=1.6, 8.4 Hz, 1H), 7.72 (d,J=1.6 Hz, 1H), 11.35 (s, 1H), 13.03 (s, 1H). EIMS m/z 248 (M+1).

Preparation of4-Hydroxy-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carboxylic acidcyclohexylamide (Compound 104)

Cyclohexylamine (13.88 mL, 121.33 mmol) was added to a solution ofCompound 102 (10 g, 40.44 mmol) in toluene (200 mL) and refluxed for 4h. The solution was cooled and the solvent was evaporated under vacuum.The residue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 12 g(98%) of white solids. M.P. 269° C. ¹H NMR (DMSO-d₆): δ 1.28 (m, 5H),1.32 (m, 1H), 1.67 (m, 2H), 1.88 (m, 2H), 2.37 (s, 3H), 3.90 (m, 1H),7.26 (d, J=8.7 Hz, 1H), 7.51 (d, J=8.7 Hz, 1H), 7.74 (s, 1H), 10.49 (s,1H), 11.80 (s, 1H). EIMS m/z 301 (M+1).

Preparation of 2,4-Dichloro-6-methyl-quinoline-3-carbonitrile (Compound105)

A solution of Compound 104 (12 g, 39.95 mmol) in 40 mL neat phosphorusoxychloride was heated at 90° C. for 3 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 7.2 g (76%) of whitesolids. M.P. 159° C. ¹H NMR (DMSO-d₆): δ 2.28 (s, 3H), 7.93 (dd, J=2.0,8.8 Hz, 1H), 8.02 (d, J=8.8 Hz, 1H), 8.07 (s, 1H). EIMS m/z 237 (M+1).

Preparation of4-Chloro-6-methyl-2-oxo-1,2-dihydro-quinoline-3-carbonitrile (Compound106)

Ammonium acetate was added to a suspension of Compound 105 (7.1 g, 29.95mmol) in glacial acetic acid and heated at 140° C. for 4 h. The solutionwas cooled and poured into ice water. The solids formed were filtered,washed by water, and dried under vacuum to yield 4.5 g (69%) of whitesolids. M.P. 264° C. ¹H NMR (DMSO-d₆): δ 2.32 (s, 3H), 7.34 (d, J=8.4Hz, 1H), 7.63 (dd, J=1.5, 8.4 Hz, 1H), 7.75 (s, 1H). EIMS m/z 219 (M+1).

Preparation of6-Methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 107)

Piperazine-1-yl-thiophen-2-yl-methanone (8.73 g, 41.16 mmol) was addedto a solution of Compound 106 (4.5 g, 20.08 mmol) in toluene (50 mL) andheated overnight at 110° C. The solvent was removed under vacuum. Theresidue was suspended in water, sonicated, and filtered. The crudeproduct was purified by flash chromatography eluting with 0–2% methanolin dichloromethane gradient to yield 7.0 g (92%) of white solids. M.P.269° C. ¹H NMR (DMSO-d₆): δ 2.34 (s, 3H), 3.65 (m, 4H), 3.92 (m, 4H),7.14 (m, 1H), 7.23 (d, J=8.4 Hz, 1H), 7.43 (d, J=8.6 Hz, 1H), 7.49 (d,J=3.5 Hz, 1H), 7.51 (s, 1H), 7.81 (d, J=4.8 Hz, 1H). EIMS m/z 379 (M+1).

Preparation of1-(2-Dimethylamino-ethyl)-6-methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 108)

A solution of Compound 107 (800 mg, 2.11 mmol), 2-dimethylamino ethylchloride hydrochloride (1.52 g, 10.55 mmol), and potassium carbonate(2.91 g, 21.10 mmol) in DMF was heated overnight at 90° C. The solutionwas cooled and poured into ice water. The solids formed were filtered,washed by water, and dried. The crude product was purified by flashchromatography eluting with 0–10% methanol in ethylacetate gradient toyield 210 mg (24%) of pale yellow solids. M.P. 132° C. ¹H NMR (DMSO-d₆):δ 2.24 (s, 6H), 2.40 (s, 3H), 2.44 (m, 2H), 3.63 (m, 4H), 3.97 (m, 4H),4.29 (m, 2H), 7.17 (dd, J=3.4, 4.8 Hz, 1H), 7.49 (m, 2H), 7.57 (m, 1H),7.69 (s, 1H), 7.80 (d, J=5.2 Hz, 1H). EIMS m/z 450 (M+1). Anal.(C₂₄H₂₇N₅O₂S) C, H, N.

Preparation of6-Methyl-1-(2-morpholin-4-yl-ethyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 109)

A solution of Compound 107 (1 g, 2.64 mmol), 4-(2-chloro ethyl)morpholine (2.45 g, 13.2 mmol), and potassium carbonate (3.64 g, 26.4mmol) in DMF was heated overnight at 90° C. The solution was cooled andpoured into ice water. The solids formed were filtered, washed by water,and dried. The crude product was purified by flash chromatographyeluting with 0–10% methanol in ethylacetate gradient to yield 223 mg(17%) of white solids. M.P. 207° C. ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H),3.54 (m, 4H), 3.63 (m, 4H), 3.92 (m, 4H), 4.35 (m, 2H), 7.16 (dd, J=3.4,4.8 Hz, 1H), 7.49 (m, 2H), 7.61 (m, 1H), 7.69 (s, 1H), 7.80 (d, J=5.2Hz, 1H). EIMS m/z 492 (M+1). Anal. (C₂₆H₂₉N₅O₃S) C, H, N.

Preparation of1-(2-Dimethylamino-ethyl)-6-methyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 110)

A solution of Compound 107 (1 g., 2.64 mmol), 2-(diisopropylamino) ethylchloride hydrochloride (2.64 g, 13.2 mmol), and potassium carbonate(3.64 g, 26.4 mmol) in DMF was heated overnight at 90° C. The solutionwas cooled and poured into ice water. The solids formed were filtered,washed by water, and dried. The crude product was purified by flashchromatography eluting with 0–10% methanol in ethylacetate gradient toyield 409 mg (30%) of pale yellow solids. M.P. 110–117° C. ¹H NMR(DMSO-d₆): δ 0.93 (s, 12H), 2.40 (s, 3H), 2.59 (m, 2H), 3.01 (m, 2H),3.61 (m, 4H), 3.91 (m, 4H), 4.15 (m, 2H), 7.16 (dd, J=3.4, 4.8 Hz, 1H),7.49 (m, 2H), 7.58 (m, 1H), 7.69 (s, 1H), 7.80 (d, J=5.2 Hz, 1H). EIMSm/z 506 (M+1). Anal. (C₂₈H₃₅N₅O₂S) C, H, N.

Preparation of6-Methyl-2-oxo-1-(2-pyrrolidin-1-yl-ethyl)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 111)

A solution of Compound 107 (1 g., 2.64 mmol), 1-(2-chloroethyl)pyrrolidine hydrochloride (2.24 g, 13.2 mmol), and potassium carbonate(3.64 g, 26.4 mmol) in DMF was heated overnight at 90° C. The solutionwas cooled and poured into ice water. The solids formed were filtered,washed by water, and dried. The crude product was purified by flashchromatography eluting with 0–10% methanol in ethylacetate gradient toyield 236 mg (19%) of pale yellow solids. M.P. 139–143° C. ¹H NMR(DMSO-d₆): δ 1.67 (m, 4H), 2.41 (s, 3H), 2.53 (m, 4H), 2.61 (m, 2H),3.01 (m, 2H), 3.62 (m, 4H), 3.93 (m, 4H), 4.30 (m, 2H), 7.16 (dd, J=3.4,4.8 Hz, 1H), 7.49 (m, 2H), 7.60 (m, 1H), 7.69 (s, 1H), 7.80 (d, J=5.2Hz, 1H). EIMS m/z 476 (M+1). Anal. (C₂₆H₂₉N₅O₂S) C, H, N.

Preparation of 4-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acidethyl ester (Compound 112)

Neat diethyl malonate (18.05 g, 112.7 mmol) was added slowly to asuspension of sodium hydride (60% in mineral oil, 4.96 mg, 124 mmol) indimethylacetamide under N₂ atmosphere. The mixture was stirred at roomtemperature until the evolution of hydrogen gas ceased, then the mixturewas heated to 90° C. for 30 min. and cooled to room temperature. Asolution of isatoic anhydride (20 g, 124 mmol) in dimethylacetamide wasadded slowly and the mixture heated overnight at 110° C. The mixture wascooled to room temperature, poured into ice water, and acidified by cold10% HCl. The solids formed were filtered and washed several times bywater to yield 8.7 g (30%) of white solids. M.P. 173° C. ¹H NMR(DMSO-d₆): δ 1.31 (t, J=6.6 Hz, 3H), 4.34 (q, J=6.6 Hz, 2H), 7.20 (t,J=8.0 Hz, 1H), 7.27 (d, J=8.4 Hz, 1H), 7.62 (t, J=7.2 Hz, 1H), 7.93 (d,J=8.0 Hz, 1H), 11.50 (b, 1H); EIMS: 234 (M+H).

Preparation of 4-Hydroxy-2-oxo-1,2-dihydro-quinoline-3-carboxylic acidcyclohexylamide (Compound 113)

Cyclohexylamine (13.88 mL, 121.33 mmol) was added to a solution ofCompound 112 (9.4 g, 40.44 mmol) in toluene (200 mL) and refluxed for 4h. The solution was cooled and the solvent was evaporated under vacuum.The residue obtained was suspended in water, briefly sonicated, andfiltered. The crude product was recrystallized by ether to yield 10.1 g(87%) of white solids. M.P. 223° C. ¹H NMR (DMSO-d₆): δ 1.3 (m, 4H), 1.6(m, 2H), 1.7 (m, 2H), 1.9 (m, 2H), 3.8 (m, 1H), 7.25 (t, J=7.7 Hz, 1H),7.35 (d, J=8.3 Hz, 1H), 7.65 (t, J=7.2 Hz, 1H), 7.95 (d, J=7.7 Hz, 1H),10.44 (b, 1H); EIMS (neg. mode): 285 (M−H).

Preparation of 2,4-Dichloro-quinoline-3-carbonitrile (Compound 114)

A solution of Compound 113 (8.5 g, 30 mmol) in 40 mL neat phosphorusoxychloride was heated at 90° C. for 3 h. The solvent was evaporatedunder reduced pressure. The residue was suspended in ice water andneutralized by solid sodium bicarbonate. The solids formed werefiltered, washed by water, and purified by flash chromatography elutingwith 1% methanol in dichloromethane to yield 6.0 g (91%) of whitesolids. M.P. 157° C. ¹H NMR (DMSO-d₆): δ ¹H-NMR (DMSO-d₆): 7.9 (m, 1H),8.09 (d, J=4.3 Hz, 2H), 8.28 (d, J=8.8 Hz, 1H); EIMS: 223 (M+H).

Preparation of 4-Chloro-2-oxo-1,2-dihydro-quinoline-3-carbonitrile(Compound 115)

Ammonium acetate (2.1 g, 27 mmol) was added to a suspension of Compound114 (6.0 g, 27 mmol) in glacial acetic acid and heated at 140° C. for 4h. The solution was cooled and poured into ice water. The solids formedwere filtered, washed by water, and dried under vacuum to yield 5.2 g(94%) of white solids. M.P. 302° C. ¹H NMR (DMSO-d₆): δ ¹H-NMR(DMSO-d₆): 7.4 (m, 2H), 7.79 (t, J=7.6 Hz, 1H), 7.96 (d, J=8.0 Hz, 1H);EIMS: 205 (M+H).

Preparation of2-Oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 116)

Piperazine-1-yl-thiophen-2-yl-methanone (8.0 g, 41 mmol) was added to asolution of Compound 115 (4.1 g, 20 mmol) in toluene (50 mL) and heatedovernight at 110° C. The solvent was removed under vacuum. The residuewas suspended in water, sonicated, and filtered. The crude product waspurified by flash chromatography eluting with 0–2% methanol indichloromethane gradient to yield 6.4 g (88%) of white solids. M.P. 264°C. ¹H NMR (DMSO-d₆): δ 3.7 (m, 4H), 3.9 (m, 4H), 7.16 (t, J=4.8 Hz, 1H),7.23 (t, J=5.9 Hz, 1H), 7.31 (d, J=8.6 Hz, 1H), 7.49 (d, J=4.0 Hz, 1H),7.61 (t, J=8.3 Hz, 1H), 7.8 (m, 2H), 11.90 (b, 1H); EIMS: 364 (M+H).

Preparation of Alkylation at N-1 Position of Quinolinone Moiety

The compounds referred to as Compound 117–158 were prepared by applyingeither General Procedure C or General Procedure D.

General Procedure C

A solution of Compound 116 (364 mg, 1 mmol) and potassium carbonate (691g, 5 mmol) with 2.5 mmol of the corresponding alkyl halide (chloro,bromo or iodo) in DMF was heated overnight at 90° C. The solution wascooled and poured into ice water. The solids formed were filtered,washed by water, and dried. In cases where solids were not formed, theproduct was extracted with either dichloromethane or n-butanol andconcentrated under vacuum. The crude product was purified by flashchromatography eluting with 0–5% methanol in dichloromethane gradient.

General Procedure D

A solution of Compound 116 (364 mg, 1 mmol) in DMF was added to astirred suspension of NaH (60% in mineral oil, 44 mg, 1.1 mmol) in DMFat room temperature under argon atmosphere. The solution was stirred atroom temperature for 1 h and the corresponding alkyl halide was addedvia syringe. The solution was further stirred at room temperature for 3to 48 h (TLC control). The reaction was worked up as described inGeneral Procedure C.

Preparation of1-(2-Dimethylamino-ethyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 117)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure D to yield white solids. Yield 211 mg (48%). M.P.96–99° C. ¹H NMR (DMSO-d₆): δ 2.20 (s, 6H), 2.46 (m, 1H), 2.71 (s, 1H),3.68 (m, 4H), 3.93 (m, 4H), 4.30 (m, 1H), 4.55 (m, 1H), 7.16 (dd, J=3.4,4.8 Hz, 1H), 7.32 (m, 1H), 7.49 (m, 1H), 7.59 (d, J=8.4 Hz, 1H), 7.71(m, 2H), 8.02 (d, J=5.2 Hz, 1H). EIMS m/z 436 (M+1). Anal. (C₂₃H₂₅N₅O₂S)C, H, N.

Preparation of1-Isobutyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 118)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure D to yield white solids. Yield 89 mg (21%). M.P.209° C. ¹H NMR (DMSO-d₆): δ 0.89 (s, 6H), 2.12 (m, 1H), 3.65 (m, 4H),3.93 (m, 4H), 4.11 (d, J=7.6 Hz, 2H), 7.16 (dd, J=3.6, 4.8 Hz, 1H), 7.32(m, 1H), 7.49 (dd, J=0.8, 3.6 Hz, 1H), 7.60 (d, J=8.8 Hz, 1H), 7.70 (m,1H), 7.79 (dd, J=1.2, 4.8 Hz, 1H), 7.93 (dd, J=1.2, 8.0 Hz, 1H). EIMSm/z 421 (M+1). Anal. (C₂₃H₂₄N₄O₂S) C, H, N.

Preparation of1-(4-Methoxybenzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 119)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure D to yield white solids. Yield 127 mg (26%). M.P.246° C. ¹H NMR (DMSO-d₆): δ 3.68 (m, 4H), 3.70 (s, 3H), 3.93 (m, 4H),5.41 (s, 2H), 6.86 (m, 2H), 7.16 (m, 3H), 7.29 (m, 1H), 7.49 (m, 2H),7.63 (m, 1H), 7.80 (d, J=4.8, 2H), 7.93 (d, J=5.2 Hz, 1H). EIMS m/z 485(M+1). Anal. (C₂₇H₂₄N₄O₃S) C, H, N.

Preparation of1-(2-Cyclohexyl-ethyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 120)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. MP: 213–215° C.; ¹H-NMR (DMSO-d₆): 0.9 (m, 2H),1.2 (m, 3H), 1.4 (m, 3H), 1.6 (m, 3H), 1.7 (m, 2H), 3.6 (m, 4H), 3.92(s, 4H), 4.22 (t, J=7.2 Hz, 2H), 7.2 (m, 1H), 7.33 (t, J=7.6 Hz, 1H),7.49 (dd, J=0.8, 3.6 Hz, 1H), 7.55 (d, J=8.4 Hz, 1H), 7.7 (m, 1H), 7.81(dd, J=1.2, 5.2 Hz, 1H), 7.93 (dd, J=1.2, 8.0 Hz, 1H); EIMS: 498 (M+Na).

Preparation of2-(2-Cyclohexyl-ethoxy)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 121)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. MP: 170–173° C.; ¹H-NMR (DMSO-d₆): 1.0 (m, 2H),1.2 (m, 3H), 1.5 (m, 1H), 1.7 (m, 7H), 3.7 (m, 4H), 3.94 (b, 4H), 4.48(t, J=6.8 Hz, 2H), 7.2 (m, 1H), 7.4 (m, 1H), 7.50 (dd, J=1.2, 3.6 Hz,1H), 7.7 (m, 2H), 7.81 (dd, J=0.8, 4.8 Hz, 1H), 8.01 (d, J=8.4 Hz, 1H);EIMS: 475 (M+H).

Preparation of2-Oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1-(4-trifluoromethyl-benzyl)-1,2-dihydro-quinoline-3-carbonitrile(Compound 122)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. MP: 248° C.; ¹H-NMR (DMSO-d₆): 3.72 (s, 4H),3.95 (s, 4H), 5.59 (s, 2H), 7.2 (m, 1H), 7.32 (t, J=8.0 Hz, 1H), 7.39(d, J=8.4 Hz, 1H), 7.43 (d, J=8.0 Hz, 2H), 7.51 (dd, J=1.2, 3.6 Hz, 1H),7.6 (m, 1H), 7.69 (d, J=8.0 Hz, 2H), 7.82 (dd, J=1.2, 5.2 Hz, 1H), 7.97(dd, J=1.2, 8.4 Hz, 1H); EIMS: 545 (M+Na).

Preparation of1-Cyclohexylmethyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 123)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. MP: 122–127° C.; ¹H-NMR (DMSO-d₆): 1.1 (m, 5H),1.6 (m, 6H), 3.6 (m, 4H), 3.93 (b, 4H), 4.11 (b, 2H), 7.2 (m, 1H), 7.32(t, J=7.6 Hz, 1H), 7.49 (dd, J=0.8, 3.6 Hz, 1H), 7.61 (d, J=8.4 Hz, 1H),7.7 (m, 1H), 7.80 (dd, J=1.2, 5.2 Hz, 1H), 7.9 (m, 1H); EIMS: 461 (M+H).

Preparation of2-Cyclohexylmethoxy-4-[4-(thiophene-2-carbonyl)-piperazin-1]-quinoline-3-carbonitrile(Compound 124)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 166° C.; ¹H-NMR (DMSO-d₆): 1.4 (m, 5H), 1.7(m, 6H), 3.68 (b, 4H), 3.94 (b, 4H), 4.25 (d, J=6.0 Hz, 2H), 7.2 (m,1H), 7.4 (m, 1H), 7.50 (dd, J=0.8, 3.6 Hz, 1H), 7.7 (m, 2H), 7.81 (dd,J=1.2, 5.2 Hz, 1H), 8.01 (d, 8.0 Hz, 1H); EIMS: 461 (M+H).

Preparation of2-Oxo-1-phenethyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 125)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 258–260° C.; ¹H-NMR (DMSO-d₆): 2.8 (m, 2H),3.65 (b, 4H), 3.93 (b, 4H), 4.4 (m, 2H), 7.2 (m, 1H), 7.3 (m, 6H), 7.50(dd, J=1.2, 3.6 Hz, 1H), 7.7 (m, 2H), 7.81 (dd, J=1.2, 5.2 Hz, 1H), 7.95(dd, J=1.2, 8.4 Hz, 1H); EIMS: 469 (M+H).

Preparation of2-Phenethyloxy-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 126)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 145–147° C.; ¹H-NMR (DMSO-d₆): 3.09 (t,J=6.8 Hz, 2H), 3.68 (b, 4H), 3.93 (b, 4H), 4.62 (t, J=6.8 Hz, 2H), 7.1(m, 1H), 7.2 (m, 1H), 7.31 (t, J=7.2 Hz, 2H), 7.4 (m, 2H), 7.5 (m, 1H),7.51 (dd, J=1.2, 3.6 Hz, 1H), 7.7 (m, 2H), 7.81 (dd, J=0.8, 4.8 Hz, 1H),8.01 (d, J=8.4 Hz, 1H); EIMS: 469 (M+H).

Preparation of2-(4-Methoxy-benzyloxy)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 127)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 192–194° C.; ¹H-NMR (DMSO-d₆): 3.69 (b,4H), 3.76 (s, 3H), 3.94 (b, 4H), 5.47 (s, 2H), 6.96 (d, J=8.4 Hz, 2H),7.2 (m, 1H), 7.5 (m, 4H), 7.78 (d, J=3.6, 2H), 7.81 (dd, J=1.2, 5.2 Hz,1H), 8.03 (d, J=8.4 Hz, 1H); EIMS: 485 (M+H).

Preparation of2-Oxo-1-(2-oxo-2-phenyl-ethyl)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 128)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 260–261° C.; ¹H-NMR (DMSO-d₆): 3.71 (b,4H), 3.96 (b, 4H), 5.90 (s, 2H), 7.17 (m, 1H), 7.33 (m, 1H), 7.45 (d,J=8.8 Hz, 1H), 7.51 (dd, J=1.2, 4.0 Hz, 1H), 7.6 (m, 3H), 7.75 (t, J=7.6Hz, 1H), 7.81 (dd, J=0.8, 4.8 Hz, 1H), 7.97 (dd, J=1.2, 8.0 Hz, 1H),8.14 (d, J=7.2 Hz, 2H); EIMS: 483 (M+H).

Preparation of1-Naphthalen-2-ylmethyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 129)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 276–279° C.; ¹H-NMR (DMSO-d₆): 3.71 (b,4H), 3.96 (b, 4H), 5.65 (b, 2H), 7.2 (m, 1H), 7.3 (m, 1H), 7.41 (dd,J=1.6, 8.4 Hz, 1H), 7.5 (m, 3H), 7.51 (dd, J=1.2, 3.6 Hz, 1H), 7.6 (m,1H), 7.7 (s, 1H), 7.8 (m, 2H), 7.9 (m, 2H), 7.96 (d, J=9.2 Hz, 1H);EIMS: 505 (M+H).

Preparation of2-(Naphthalen-2-ylmethoxy)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 130)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 250° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 4H),3.95 (b, 4H), 5.72 (s, 2H), 7.2 (m, 1H), 7.5 (m, 4H), 7.67 (dd, J=1.6,8.4 Hz, 1H), 7.8 (m, 3H), 7.9 (m, 3H), 8.1 (m, 2H); EIMS: 505 (M+H).

Preparation of1-(3-Dimethylamino-propyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 131)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 203–204° C.; ¹H-NMR (DMSO-d₆): 1.7 (m, 2H),2.24 (s, 6H), 2.4 (m, 2H), 3.64 (b, 4H), 3.93 (b, 4H), 4.23 (t, J=7.2Hz, 2H), 7.2 (m, 1H), 7.34 (m, 1H), 7.50 (dd, J=1.2, 3.6 Hz, 1H), 7.67(d, J=8.4 Hz, 1H), 7.7 (m, 1H), 7.81 (dd, J=1.2, 5.2 Hz, 1H), 7.95 (dd,J=1.2, 8.0 Hz, 1H); EIMS: 450 (M+H).

Preparation of2-(3-Dimethylamino-propoxy)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 132)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 130° C.; ¹H-NMR (DMSO-d₆): 1.9 (m, 2H),2.17 (s, 6H), 2.43 (t, J=6.8 Hz, 2H), 3.7 (m, 4H), 3.95 (b, 4H), 4.47(t, J=6.8 Hz, 2H), 7.2 (m, 1H), 7.5 (m, 1H), 7.52 (dd, J=0.8, 3.6 Hz,1H), 7.7 (m, 2H), 7.81 (dd, J=1.2, 5.2 Hz, 1H), 8.02 (d, J=8.0 Hz, 1H);EIMS: 450 (M+H).

Preparation of1-(2,2-Dimethyl-propyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 133)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 111–116° C.; ¹H-NMR (DMSO-d₆): 0.9 (s, 9H),3.65 (b, 4H), 3.93 (b, 4H), 4.20 (b, 2H), 7.2 (m, 1H), 7.30 (t, J=7.6Hz, 1H), 7.50 (dd, J=1.2, 5.2 Hz, 1H), 8.02 Hz, 1H), 7.7 (m, 1H), 7.8(m, 2H), 7.91 (dd, J=1.6, 8.4 Hz, 1H); EIMS: 457 (M+Na).

Preparation of2-Oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1-(2,2,2-trifluoro-ethyl)-1,2-dihydro-quinoline-3-carbonitrile(Compound 134)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 242° C.; ¹H-NMR (DMSO-d₆): 3.71 (b, 4H),3.93 (b, 4H), 5.2 (m, 2H), 7.2 (m, 1H), 7.4 (m, 1H), 7.50 (dd, J=0.8,3.6 Hz, 1H), 7.7 (m, 2H), 7.81 (dd, J=0.8, 4.8 Hz, 1H), 7.95 (d, J=8.0Hz, 1H); EIMS: 447 (M+H).

Preparation of4-[4-(Thiophene-2-carbonyl)-piperazin-1-yl]-2-(2,2,2-trifluoro-ethoxy)-quinoline-3-carbonitrile(Compound 135)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 204° C.; ¹H-NMR (DMSO-d₆): 3.70 (b, 4H),3.95 (b, 4H), 5.2 (m, 2H), 7.2 (m, 1H), 7.5 (m, 2H), 7.8 (m, 3H), 8.1(m, 1H); EIMS: 447 (M+H).

Preparation of2-Oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1-(4-trifluoromethoxy-benzyl)-1,2-dihydro-quinoline-3-carbonitrile(Compound 136)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 150–160° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 4H),3.9 (m, 4H), 5.52 (s, 2H), 7.2 (m, 1H), 7.3 (m, 5H), 7.44 (d, J=8.3 Hz,1H), 7.51 (d, J=3.8 Hz, 1H), 7.64 (t, J=7.1 Hz, 1H), 7.81 (dd, J=1.0,5.0 Hz, 1H), 7.96 (J=1.3, 8.3 Hz, 1H); EIMS: 539 (M+H).

Preparation of4-[4-(Thiophene-2-carbonyl)-piperazin-1-yl]-2-(4-trifluoromethoxy-benzyloxy)-quinoline-3-carbonitrile (Compound 137)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 170–172° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 4H),3.98 (b, 4H), 5.63 (s, 2H), 7.2 (m, 1H), 7.46 (d, J=8.0 Hz, 2H), 7.5 (m,2H), 7.70 (d, J=8.8 Hz, 2H), 7.8 (m, 2H), 7.85 (dd, J=0.8, 4.8 Hz, 1H),8.08 (d, 8.4 Hz, 1H); EIMS: 539 (M+H).

Preparation of1-(3-Fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 138)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 261–263° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 4H),3.94 (b, 4H), 5.50 (s, 2H), 7.1 (m, 3H), 7.2 (m, 1H), 7.3 (m, 2H), 7.41(d, J=8.4 Hz, 1H), 7.51 (dd, J=0.8, 3.6 Hz, 1H), 7.6 (m, 1H), 7.81 (dd,J=1.2, 5.2 Hz, 1H), 7.9 (m, 1H); EIMS: 437 (M+H).

Preparation of1-(2-Fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 139)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 203–205° C.; ¹H-NMR (DMSO-d₆): 3.7 (m, 4H),3.9 (m, 4H), 5.50 (s, 2H), 6.86 (t, J=6.7 Hz, 1H), 7.07 (t, J=7.5 Hz,1H), 7.2 (m, 1H), 7.3 (m, 4H), 7.50 (dd, J=0.9, 3.6 Hz, 1H), 7.7 (m,1H), 7.81 (dd, J=0.9, 5.0 Hz, 1H), 7.97 (dd, J=1.2, 8.2 Hz, 1H); EIMS:473 (M+H).

Preparation of2-Oxo-1-propyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 140)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 210–212° C.; ¹H-NMR (DMSO-d₆): 0.97 (t,J=7.2 Hz, 3H), 1.6 (m, 2H), 3.6 (m, 4H), 3.9 (m, 4H), 4.16 (t, J=7.6 Hz,2H), 7.1 (m, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.49 (dd, J=0.8, 3.6 Hz, 1H),7.63 (d, J=8.4 Hz, 1H), 7.7 (m, 1H), 7.79 (dd, J=1.2, 5.2 Hz, 1H), 7.94(dd, J=1.2, 8.4 Hz, 1H); EIMS: 407 (M+H).

Preparation of2-Propoxy-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 141)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 198–199° C.; ¹H-NMR (DMSO-d₆): 1.01 (t,J=7.6 Hz, 3H), 1.8 (m, 2H), 3.69 (b, 4H), 3.94 (b, 4H), 4.41 (t, J=6.4Hz, 2H), 7.2 (m, 1H), 7.4 (m, 1H), 7.50 (d, J=3.6 Hz, 1H), 7.7 (m, 2H),7.80 (d, J=4.8 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H), EIMS: 407 (M+H).

Preparation of1-Butyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 142)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 155–157° C.; ¹H-NMR (DMSO-d₆): 0.93 (t,J=7.2 Hz, 3H), 1.4 (m, 2H), 1.6 (m, 2H), 3.6 (m, 4H), 3.92 (b, 4H), 4.20(t, J=7.6 Hz, 2H), 7.2 (m, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.50 (d, J=3.2Hz, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.75 (t, J=7.2 Hz, 1H), 7.80 (d, J=5.2Hz, 1H), 7.94 (d, J=7.6 Hz, 1H); EIMS: 421 (M+H).

Preparation of2-Butoxy-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 143)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 190–191° C.; ¹H-NMR (DMSO-d₆): 0.96 (t,J=7.2 Hz, 3H), 1.5 (m, 2H), 1.8 (m, 2H), 3.68 (b, 4H), 3.94 (b, 4H),4.46 (t, J=6.4 Hz, 2H), 7.2 (m, 1H), 7.4 (m, 1H), 7.50 (dd, J=0.8, 3.6Hz, 1H), 7.7 (m, 2H), 7.80 (dd, J=1.2, 5.2 Hz, 1H), 8.02 (d, J=8.4 Hz,1H); EIMS: 421 (M+H).

Preparation of1-(3-Hydroxy-propyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 144)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 209–210° C.; ¹H-NMR (DMSO-d₆): 1.7 (m, 2H),3.5 (m, 2H), 3.6 (m, 4H), 3.9 (m, 4H), 4.26 (t, J=7.2 Hz, 2H), 4.65 (t,J=5.2 Hz, 1H), 7.1 (m, 1H), 7.34 (t, J=7.2 Hz, 1H), 7.49 (d, J=3.2 Hz,1H), 7.64 (d, J=8.8 Hz, 1H), 7.76 (t, J=7.2 Hz, 1H), 7.80 (d, J=5.2 Hz,1H), 7.94 (d, J=7.6 Hz, 1H); EIMS: 423 (M+H).

Preparation of1-Cyclopropylmethyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 145)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 198° C.; ¹H-NMR (DMSO-d₆): 0.45 (d, J=6.4Hz, 4H), 1.2 (m, 1H), 3.6 (m, 4H), 3.9 (m, 4H), 4.17 (d, J=6.8 Hz, 2H),7.1 (m, 1H), 7.3 (m, 1H), 7.49 (dd, J=1.2, 3.6 Hz, 1H), 7.7 (m, 2H),7.79 (dd, J=1.2, 4.8 Hz, 1H), 7.94 (d, J=7.6 Hz, 1H); EIMS: 419 (M+H).

Preparation of2-Cyclopropylmethoxy-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 146)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 200–203° C.; ¹H-NMR (DMSO-d₆): 0.5 (m, 2H),0.6 (m, 2H), 1.3 (m, 1H), 3.69 (b, 4H), 3.94 (b, 4H), 4.31 (d, J=6.8 Hz,2H), 7.2 (m, 1H), 7.5 (m, 1H), 7.49 (dd, J=0.8, 3.6 Hz, 1H), 7.7 (m,2H), 7.8 (dd, J=0.8, 4.8 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H); EIMS: 419(M+H).

Preparation of1-(4-Cyano-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 147)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 260–263° C.; ¹H-NMR (DMSO-d₆): 3.72 (b,4H), 3.95 (b, 4H), 5.58 (s, 2H), 7.2 (m, 1H), 7.31 (t, J=7.6 Hz, 1H),7.34 (d, J=8.8 Hz, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.50 (dd, J=0.8, 3.6 Hz,1H), 7.63 (t, J=7.2 Hz, 1H), 7.8 (m, 3H), 7.96 (d, J=7.2 Hz, 1H); EIMS:480 (M+H).

Preparation of2-(4-Cyano-benzyloxy)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 148)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 214–218° C.; ¹H-NMR (DMSO-d₆): 3.71 (b,4H), 3.95 (b, 4H), 5.58 (s, 2H), 7.17 (t, J=2.8 Hz, 1H), 7.31 (t, J=7.2Hz, 1H), 7.35 (d, J=8.8 Hz, 1H), 7.40 (d, J=8.0 Hz, 2H), 7.50 (d, J=3.6Hz, 1H), 7.63 (t, J=7.2 Hz, 1H), 7.8 (m, 3H), 7.96 (d, J=8.0 Hz, 1H);EIMS: 480 (M+H).

Preparation of2-Oxo-1-pentyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 149)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 201–202° C.; ¹H-NMR (DMSO-d₆): 0.88 (t,J=6.8 Hz, 3H), 1.3 (m, 4H), 1.6 (m, 2H), 3.6 (m, 4H), 3.92 (b, 4H), 4.19(t, J=7.6 Hz, 2H), 7.2 (m, 1H), 7.33 (t, J=7.6 Hz, 1H), 7.49 (dd, J=0.8,3.6 Hz, 1H), 7.61 (d, J=8.4 Hz, 1H), 7.75 (t, J=7.2 Hz, 1H), 7.8 (dd,J=0.8, 4.8 Hz, 1H), 7.94 (dd, J=0.8, 8.0 Hz, 1H); EIMS: 435 (M+H).

Preparation of2-Pentyloxy-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 150)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 152–155° C.; ¹H-NMR (DMSO-d₆): 0.91 (t,J=7.2 Hz, 3H), 1.4 (m, 4H), 1.7 (m, 2H), 3.7 (m, 4H), 3.94 (b, 4H), 4.45(t, J=6.8 Hz, 2H), 7.17 (t, J=4.4 Hz, 1H), 7.4 (m, 1H), 7.50 (d, J=3.6Hz, 1H), 7.7 (m, 2H), 7.80 (d, J=4.8 Hz, 1H), 8.02 (d, J=8.4 Hz, 1H);EIMS: 435 (M+H).

Preparation of1-(4-Methyl-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinoline-3-carbonitrile(Compound 151)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 236–238° C.; ¹H-NMR (DMSO-d₆): 2.25 (s,3H), 3.7 (m, 4H), 3.9 (m, 4H), 5.44 (s, 2H), 7.12 (s, 4H), 7.2 (m, 1H),7.29 (t, J=7.8 Hz, 1H), 7.41 (d, J=8.4 Hz, 1H), 7.50 (dd, J=1.2, 4.0 Hz,1H), 7.6 (m, 1H), 7.81 (dd, J=0.8, 4.8 Hz, 1H), 7.94 (dd, J=1.2, 8.4 Hz,1H); EIMS: 469 (M+H).

Preparation of2-(4-Methyl-benzyloxy)-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-quinoline-3-carbonitrile(Compound 152)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 188–191° C.; ¹H-NMR (DMSO-d₆): 2.31 (s,3H), 3.7 (m, 4H), 3.9 (m, 4H), 5.50 (s, 2H), 7.2 (m, 1H), 7.21 (d, J=7.6Hz, 2H), 7.41 (d, J=8.0 Hz, 2H), 7.5 (m, 2H), 7.77 (d, J=4.0 Hz, 2H),7.80 (dd, J=1.2, 5.2 Hz, 1H), 8.03 (d, J=8.4 Hz, 1H); EIMS: 469 (M+H).

Preparation of2-Oxo-1-propyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carboxylicacid ethyl ester (Compound 153)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 92–96° C.; ¹H-NMR (DMSO-d₆): 0.91 (t, J=7.2Hz, 3H), 1.30 (t, J=7.2 Hz, 3H), 1.6 (m, 2H), 3.13 (s, 4H), 3.88 (s,4H), 4.3 (m, 4H), 7.1 (m, 1H), 7.4 (m, 1H), 7.45 (dd, J=1.2, 3.6 Hz,1H), 7.79 (dd, J=0.8, 4.8 Hz, 1H), 8.34 (dd, J=1.6, 8.0 Hz, 1H), 8.70(dd, J=1.6, 4.4 Hz, 1H); EIMS: 455 (M+H).

Preparation of1-Butyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carboxylicacid ethyl ester (Compound 154)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 90–96° C.; ¹H-NMR (DMSO-d₆): 0.92 (t, J=7.2Hz, 3H), 1.3 (m, 5H), 1.6 (m, 2H), 3.13 (s, 4H), 3.88 (s, 4H), 4.32 (m,4H), 7.15 (m, 1H), 7.38 (m, 1H), 7.45 (dd, J=1.2, 3.6, 1H), 7.79 (dd,J=1.2, 5.2 Hz, 1H), 8.34 (dd, J=1.6, 8.0 Hz, 1H), 8.70 (dd, J=1.6, 4.4Hz, 1H); EIMS: 469 (M+H).

Preparation of1-Allyl-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carboxylicacid ethyl ester (Compound 155)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 89–96° C.; ¹H-NMR (DMSO-d₆): 1.30 (t, J=7.2Hz, 3H), 3.15 (s, 4H), 3.89 (s, 4H), 4.31 (t, J=7.2 Hz, 2H), 5.0 (m,4H), 5.9 (m, 1H), 7.15 (m, 1H), 7.39 (dd, J=4.8, 8.0 Hz, 1H), 7.46 (dd,J=0.8, 3.6 Hz), 7.79 (dd, J=0.8, 4.8 Hz, 1H), 8.34 (dd, J=1.6, 8.0 Hz,1H), 8.68 (dd, J=1.6, 4.8 Hz, 1H); EIMS: 453 (M+H).

Preparation of1-(2-Fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carboxylicacid ethyl ester (Compound 156)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 105–110° C.; ¹H-NMR (DMSO-d₆): 1.29 (t,J=7.2 Hz, 3H), 3.19 (s, 4H), 3.91 (s, 4H), 4.30 (q, J=7.2 Hz, 2H), 5.60(s, 2H), 6.81 (m, 1H), 7.04 (m, 1H), 7.2 (m, 3H), 7.39 (m, 1H), 7.46(dd, J=0.8, 3.6 Hz, 1H), 7.80 (dd, J=0.8, 4.8 Hz, 1H), 8.38 (dd, J=1.6,8.0 Hz, 1H), 8.63 (dd, J=1.6, 4.4 Hz, 1H); EIMS: 521 (M+H).

Preparation of1-(3-Fluoro-benzyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carboxylicacid ethyl ester (Compound 157)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 105–110° C.; ¹H-NMR (DMSO-d₆): 1.29 (t,J=6.8 Hz, 3H), 3.18 (s, 4H), 3.89 (s, 4H), 4.31 (q, J=6.8 Hz, 2H), 5.56(s, 2H), 7.1 (m, 4H), 7.4 (m, 3H), 7.79 (d, J=4.4 Hz, 1H), 8.36 (d,J=7.6 Hz, 1H), 8.67 (d, J=3.2 Hz, 1H); EIMS: 521 (M+H).

Preparation of1-(3-Dimethylamino-propyl)-2-oxo-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-[1,8]naphthyridine-3-carboxylicacid ethyl ester (Compound 158)

The compound was prepared from the corresponding alkyl halide accordingto General Procedure C. M.P. 84–94° C.; ¹H-NMR (DMSO-d₆): 1.30 (t, J=7.2Hz, 3H), 1.7 (m, 2H), 2.14 (s, 6H), 2.30 (t, J=6.8 Hz, 2H), 3.13 (b,4H), 3.88 (b, 4H), 4.3 (m, 4H), 7.2 (m, 1H), 7.3 (m, 1H), 7.45 (dd,J=1.2, 3.6 Hz, 1H), 7.79 (dd, J=0.8, 4.8 Hz, 1H), 8.34 (dd, J=2.0, 8.0Hz, 1H), 8.70 (dd, J=2.0, 4.8 Hz, 1H); EIMS: 498 (M+H).

Preparation of2-Oxo-1-phenyl-4-[4-(thiophene-2-carbonyl)-piperazin-1-yl]-1,2-dihydro-quinolin-3-carbonitrile(Compound 159)

Cupric acetate (497 mg, 2.74 mmol), triethylamine (380 μL, 2.74 mmol),and phenyl boronic acid (335 mg, 2.74 mmol) were added successively to asolution of Compound 116 (500 mg, 1.37 mmol) in dichloromethane. Thesolution was stirred at room temperature for 48 h. The solution wasfiltered through celite and washed successively by saturated NaHCO₃solution, water, and brine. The organic phase was dried over Na₂SO₄ andevaporated to yield a residue which was purified by flash chromatographyeluting with 0–1% MeOH in dichloromethane gradient to get 187 mg (31%)of white solids. M.P. 289° C. ¹H NMR (DMSO-d₆): δ 3.78 (m, 4H), 4.02 (m,4H), 6.60 (d, J=7.6 Hz, 1H), 7.22 (m, 1H), 7.38 (m, 3H), 7.57–7.71 (m,5H), 7.88 (dd, J=0.8, 4.8 Hz, 1H), 8.02 (d, J=7.2 Hz, 1h). EIMS m/z 441(M+1). Anal. (C₂₅H₂₀N₄O₂S) C, H, N.

Example 4

The following inhibitors of MIF were prepared by the methods describedin the examples. Each of these MIF inhibitors belongs to the class ofcompounds of structure 1(a) described above. Results of tautomeraseassays indicated that each of the following candidate compounds exhibitparticularly high levels of inhibition of MIF activity. These MIFinhibitors were each active at concentrations of from 0.01 nM to 50 μM.

Example 5

The following inhibitors of MIF of preferred embodiments can be preparedby the methods described in the examples.

Example 6

Compound 200 was tested in the THP-1 Cell Assay at several differentconcentrations. Compound 200 exhibits MIF inhibitory activity, as shownin FIG. 1. Compound 200 exhibits MIF inhibitory activity

Example 7

Compound 200 was tested for in vitro tautomerase inhibitory activity atseveral different concentrations. Compound 200 exhibits MIF inhibitoryactivity, as shown in FIG. 2.

Example 8

Compound 203 was tested for in vitro tautomerase inhibitory activity atseveral different concentrations. Compound 203 exhibits MIF inhibitoryactivity, as shown in FIG. 3.

Example 9

The MIF inhibitory activity of Compound 200 and Compound 203 werecompared. Both exhibit satisfactory MIF inhibitory activity.

TABLE 2 In Vitro Activity of MIF Inhibitors (Inhibitory Concentration(IC) of 50 μm) Tautomerase Average THP-1/MIF Average Compound Assay IC50 Inhibition IC 50 200 0.30 0.32 0.023 0.15 (0.23–0.4) (0.001–0.52) (0.13–0.17) 200 0.34 — 0.15 — (0.12–0.9) (0.13–0.17) 200 — — 0.15 —(0.13–0.17) 203 0.098 0.098 — —  (0.071–0.134)

The preferred embodiments have been described in connection withspecific embodiments thereof. It will be understood that it is capableof further modification, and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practices in theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as fall within the scopeof the invention and any equivalents thereof. Each reference citedherein including but not limited to patents and technical literature, ishereby incorporated by reference in its entirety.

1. A compound having a structure:

or a stereoisomer or a pharmaceutically acceptable salt thereof,wherein: X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; R₁ is selectedfrom the group consisting of

 C₁₋₁₀ alkyl and aryl C₁₋₁₀ alkyl, wherein R₁ is unsubstituted orsubstituted with at least one substituent selected from the groupconsisting of halogen, alkoxy, alkylamino, dialkylamino, and keto; R₂and R₃ are independently selected from the group consisting of halogen,hydrogen, and C₁₋₆ alkyl; and R₇ is selected from the group consistingof cyclopentyl, phenyl, pyrazolyl, thiadiazolyl, isoxazolyl, imidazolyl,pyrrolyl, indolyl, isoquinolinyl, pyridinyl, tetrahydrothiophenyl,thienyl, furyl, tetrahydrofuranyl, thiazolidinyl, pyrazinyl,pyrrolidinyl, and piperidinyl, wherein R₇ is unsubstituted orsubstituted with at least one substituent selected from the groupconsisting of halogen, alkoxy, nitro, and alkylamino.
 2. The compound ofclaim 1, wherein X is oxygen.
 3. The compound of claim 1, wherein X issulfur.
 4. The compound of claim 1, wherein Z is —CH₂—.
 5. The compoundof claim 1, wherein Z is —C(═O)—.
 6. The compound of claim 1, wherein R₂is hydrogen and wherein R₃ is selected from the group consisting ofhydrogen, methyl, and chlorine.
 7. The compound of claim 1, wherein R₇is selected from the group consisting of


8. The compound of claim 1, wherein R₁ is —(CH₂)_(n)COOR′″, wherein n isan integer of from 1 to 4, and wherein R′″ is selected from the groupconsisting of hydrogen, fluorine, chlorine, linear C₁–C₅ alkyl, andbranched C₁–C₅ alkyl.
 9. The compound of claim 1, wherein R₁ is selectedfrom the group consisting of —(CH₂)_(n)N(R′″)₂ and—(CH₂)_(n)C(O)N(R′″)₂, wherein n is an integer of from 1 to 4, andwherein each R′″ is independently selected from the group consisting ofhydrogen, fluorine, chlorine, linear C₁–C₅ alkyl, and branched C₁–C₅alkyl.
 10. The compound of claim 1, wherein R₁ is selected from thegroup consisting of

and wherein R″″ is selected from the group consisting of hydrogen,halogen, alkyl, cyano, nitro, —COOR′″, —N(R′″)₂, —OR′″, —NHCOR′″, and—OCF₃, and wherein R′″ is independently selected from the groupconsisting of hydrogen, fluorine, chlorine, linear C₁–C₅ alkyl, andbranched C₁–C₅ alkyl.
 11. The compound of claim 1, wherein R₁ isselected from the group consisting of:


12. A composition comprising a compound of claim 1 in combination with apharmaceutically acceptable carrier or diluent.
 13. A compound having astructure:

or a stereoisomer or a pharmaceutically acceptable salt thereof,wherein: X is oxygen or sulfur; Z is —CH₂— or —C(═O)—; R₁ is selectedfrom the group consisting of

 C₁₋₁₀ alkyl and aryl C₁₋₁₀ alkyl, wherein R₁ is unsubstituted orsubstituted with at least one substituent selected from the groupconsisting of halogen, alkoxy, alkylamino, dialkylamino, and keto; R₂and R₃ are independently selected from the group consisting of halogen,hydrogen, and C₁₋₆ alkyl; and R₇ is selected from the group consistingof cyclopentyl, phenyl, pyrazolyl, thiadiazolyl, isoxazolyl, imidazolyl,pyrrolyl, indolyl, isoquinolinyl, pyridinyl, tetrahydrothiophenyl,thienyl, furyl, tetrahydrofuranyl, thiazolidinyl, pyrazinyl,pyrrolidinyl, and piperidinyl, wherein R₇ is unsubstituted orsubstituted with at least one substituent selected from the groupconsisting of halogen, alkoxy, nitro, and alkylamino.
 14. The compoundof claim 13, wherein X is oxygen.
 15. The compound of claim 13, whereinX is sulfur.
 16. The compound of claim 13, wherein Z is —CH₂—.
 17. Thecompound of claim 13, wherein Z is —C(═O)—.
 18. The compound of claim13, wherein R₂ is hydrogen and wherein R₃ is selected from the groupconsisting of hydrogen, methyl, and chlorine.
 19. The compound of claim13, wherein R₇ is selected from the group consisting of


20. The compound of claim 13, wherein R₁ is —(CH₂)_(n)COOR′″, wherein nis an integer of from 1 to 4, and wherein R′″ is selected from the groupconsisting of hydrogen, fluorine, chlorine, linear C₁–C₅ alkyl, andbranched C₁–C₅ alkyl.
 21. The compound of claim 13, wherein R₁ isselected from the group consisting of —(CH₂)_(n)N(R′″)₂ and—(CH₂)_(n)C(O)N(R′″)₂, wherein n is an integer of from 1 to 4, andwherein each R′″ is independently selected from the group consisting ofhydrogen, fluorine, chlorine, linear C₁–C₅ alkyl, and branched C₁–C₅alkyl.
 22. The compound of claim 13, wherein R₁ is selected from thegroup consisting of

and wherein R″″ is selected from the group consisting of hydrogen,halogen, alkyl, cyano, nitro, —COOR′″, —N(R′″)₂, —OR′″, —NHCOR′″, and—OCF₃, and wherein R′″ is independently selected from the groupconsisting of hydrogen, fluorine, chlorine, linear C₁–C₅ alkyl, andbranched C₁–C₅ alkyl.
 23. The compound of claim 13, wherein R₁ isselected from the group consisting of:


24. A composition comprising a compound of claim 13 in combination witha pharmaceutically acceptable carrier or diluent.